Targeted hybridization recruitment

Abstract

A tagmentation method uses binding agents and / or nucleic acid modification enzymes having hybridizable (e.g., complementary) oligonucleotides to recruit the modification enzymes to binding agents bound to their targets in chromatin. After nucleic acid modification, e.g., cleavage of nucleic acids near the binding site, and attachment of adaptors, such as amplification adaptors, tagged nucleic acids can be amplified and / or sequenced to aid in epigenetic mapping.

Description

TARGETED HYBRIDIZATION RECRUITMENTSTATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0001] None.REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the priority date of U.S. provisional application 63 / 557,976, filed February 26, 2024, the contents of which are incorporated herein in their entirety.THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

[0003] None.SEQUENCE LISTING

[0004] None.BACKGROUND

[0005] In the art of epigenetics, a number of methods for mapping post-translational modifications (PTM) on histones or other DNA binding proteins, such as transcription factors, exists. One of the leading methods is Chromatin Immunoprecipitation followed by sequencing (ChlP-seq) which requires high amount of sample input, has high background, is comprised of tedious and time-consuming steps. To circumvent some of these limitations, enzyme tethering methods for mapping histone PTMs, DNA binding proteins and other features in or associated with nucleic acids have been developed, for example using Tn5, a transposase with the ability to insert DNA sequences to alternative locations in the genome in a process called transposition. During transposition, two Tn5 monomers bind proximal, specific DNA sequences, or “transposons,” within the genome. Upon binding, dimerization between the two Tn5 monomers occurs, resulting in a complex capable of cleaving DNA. The DNA between the two bound transposons is cleaved from the chromatin and the Tn5 / DNA complex is transported to another site in the genome, where the DNA is inserted. The complex that forms after dimerization can be produced in vitro by combining Tn5 monomer with the free transposon sequence, assembling what is referred to as a “transposome”. Unlike the complex formed during the native process of transposition, transposomes are assembled with non-continuous DNA fragments. This leads to fragmentation of the DNA at the site of insertion. Additionally, the sequences of the transposons used to assemble transposomes may be customized to contain any desired sequence, in addition to the Tn5 binding sequence. The result is a protein capable of simultaneously fragmenting and inserting custom DNA sequences into chromatin, a processcalled tagmentation. A method of mapping open chromatin regions using untethered Tn5 was first described in 2013 in the scientific publication “Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position” (Buenrostro et al., 2013). Since then, Tn5 has been used in numerous other assays, including assays based on targeted tagmentation using enzyme tethering methods.

[0006] CUT&Tag refers to a targeted chromatin profiling method that uses an antibody- guided Tn5 transposase to cleave and insert sequencing adapters at specific histone modifications or protein-binding sites. Recruiting enzymes to specific genomic locations by tethering the enzyme and / or associated complex / transposon to an antibody, for the purpose of genome-wide mapping of, e.g., histone modifications and transcription factors (TF) was first described in patent applications WO 2013 / 078470 and WO 2014 / 190214 50 followed by scientific publications for CUT&RUN (Skene, P. J., and Henikoff, S., 2017) (transcription factor and histone mapping through antibody mediated recruitment of Mnase) and CUT&Tag (Kaya- Okur, H. S., et al., 2019) (histone PTM mapping through antibody mediated recruitment of the transposase Tn5).

[0007] Tn5 transposome recruiting methods such as CUT&Tag require the in situ binding of a specific antibody to a chromatin associated protein (histone PTM, TF, chromatin modifier, polymerase, etc). Then one can employ a protein-A / Tn5 fusion protein (pA-Tn5), a protein- G / Tn5 fusion protein or a protein- AG / Tn5 fusion protein that is recruited to the antibody binding location through the natural binding affinity of the protein A (pA) portion of the pA-Tn5 to the Fc region of the antibody (FIG. 1). Patent application WO 2013 / 078470 describes, among other things, direct conjugation of the Tn5 enzyme / transposome to a primary antibody, however, in practice it is understood that the primary conjugation methods used resulted in only 1-2 Tn5 transposomes conjugated to each antibody and efficient tagmentation at the antibody (or other protein) binding site requires an accumulation of Tn5 transposomes at the target site. The use of secondary antibodies and / or additional binding proteins overcomes this limitation since multiple secondary antibodies can bind to a single primary antibody and two pAs can bind to each secondary, thus resulting in many pA / Tn5 transposomes accumulating at the targeting antibody.

[0008] Similar methods are known in the art for recruiting enzymes (e.g., MNase) and associated tags and adaptors for mapping (e.g., bar codes and sequencing adaptors) to nucleic acids sites via antibody and other protein (e.g., proteins A and G) binding reactions.

[0009] The use of binding proteins such as pA and secondary antibodies imposes constraints in CUT&Tag and these other applications / methods, especially in a multiplexing applications.

[0010] The use of and differentiation between multiple primary antibodies (multiplexing) in the same reaction is only feasible when using primary antibodies from different animal species (i.e. mouse, sheep, rabbit). Secondary antibodies in those reactions are species specific, only binding to the antibody species that it was raised against. Multiplexing is possible when secondary antibodies are preincubated with barcoded pA-Tn5 or if recombinant secondaries are created as fusions to Tn5. Since primary antibodies are raised in only a limited number of animal species, this approach complicates experimental design by requiring all primary antibodies to be from different species and requiring the use of matched secondary antibodies. In practice multiplexing is maxed out at three to four targets.

[0011] Multiplexing can be accomplished without a secondary antibody by first binding the primary antibody to a pA-Tn5 transposome containing transposons with unique DNA barcodes (Golopan, S., et al., 2021).BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate exemplary embodiments and, together with the description, further serve to enable a person skilled in the pertinent art to make and use these embodiments and others that will be apparent to those skilled in the art. The invention will be more particularly described in conjunction with the following drawings wherein:

[0013] FIG. 1 shows a schematic of existing Tn5 transposome assay technologies. (FIG. 1A.) A non-targeted tagmentation assay where free Tn5 transposomes bind only at open chromatin sites is depicted. It is at these open sites where tagmentation will occur upon activation with Mg. (Fig 1B.) A targeted tagmentation assay is depicted. The primary antibody binds its target site on chromatin followed by the addition of a secondary antibody that binds the primary antibody. Next, protein A-Tn5 (pA-Tn5) is added and binds to the secondary antibody through pA affinity for the Fc region of the antibody. All unbound pA-Tn5 is washed away and tagmentation occurs only at the target location.

[0014] FIG. 2 shows a schematic of the transposome assembly with transposons that include extended single stranded regions. The single stranded 5’ overhang sequence on each transposon (transposon A and transposon B) is customizable and used for recruitment of transposomes to the oligo-conjugated antibody through hybridization. The ME (“mosaic end”) sequence is standard in the art and is conserved in all transposons. Loading Tn5 requires mixing Tn5 adapters (transposons) with Tn5 monomers at an equal molar ratio and yields one active Tn5 dimer called a transposome. The Ns represents either A, G, C or T residues within the sequence. This stretch of nucleotides is designed by the user and will be used for hybridization to recruit loaded Tn5s (transposomes). The length of the custom overhang is notrepresented by the number of N residues in the figure and could be longer or shorter than depicted.

[0015] FIG. 3 shows a conjugation strategy used to covalently attach oligonucleotide sequences to an antibody for Tn5 recruitment through hybridization. Each antibody contains multiple conjugation sites, resulting in 3 or 4 or 5 or 6 or more oligonucleotide sequences, with an average of 4 per antibody. (FIG. 3A.) Oligonucleotides can be conjugated to both antibody heavy chains. A click chemistry moiety is attached to the terminal end of the heavy chain at the sortase tag on the Abflex® antibody. For simplicity, conjugation is shown at one site on the antibody, but there are sortase tags at the terminal ends of both heavy chains. The antibody recruitment sequence containing a click chemistry moiety is reacted with the antibody to produce a primary antibody with a conjugated recruitment oligonucleotide sequence. The moiety conjugated to the AbFlex® antibody contains multiple reactive sites resulting in numerous oligonucleotide conjugations. (FIG. 3B.) A gel image shows an unconjugated AbFlex® antibody (lane 1), and a conjugated AbFlex® antibody (lane 2). The number of oligonucleotides bound to the antibody is illustrated by the arrows.

[0016] FIG. 4 shows the strategy for recruitment of T n5 transposomes to the target region using DNA hybridization. (FIG. 4A.) While the figure shows only one transposome being recruited to the antibody, it is understood that the antibody can be provided with multiple recruitment molecules, and that these can exist on either or both antibody chains. An antibody, conjugated to single stranded DNA, is bound to the target DNA binding protein and Tn5 transposomes, loaded with transposons containing the complementary sequence, are added. Hybridization occurs between the two complementary single strands of DNA and the Tn5 transposome is recruited to the target site. (FIG. 4B.) An exemplary Tn5 loading sequence (transposon) shows the design of the 5’ overhang. In this example, the sequence complementary to the antibody recruitment sequence is followed by the flow cell binding sequence (P5 / P7), a unique index, a unique molecular identifier (UMI), a read primer binding sequence required for next generation sequencing platforms (Read 1 / Read 2), and the ME sequence. The ME sequence is the only double stranded portion of the sequence. It is required for all Tn5 transposome assembly as it is where the Tn5 monomer will bind the loading sequence.

[0017] FIG. 5 shows a representative schematic of fate and function of the transposon regions. The tagmentation reaction yields a fragment of genomic DNA flanked by the partially single stranded transposons. PCR amplification with primers binding to the P5 and P7 region produces a fully double stranded library without the antibody recruitment region.

[0018] FIG. 6 shows multiplexed Tn5 transposome recruitment using DNA hybridization to three different targeting antibodies. In this example, each of Antibody 1 , Antibody 2, and Antibody 3 is uniquely conjugated with a single stranded oligonucleotide containing arecruitment sequence. The complement to that sequence is loaded into Tn5 as part of the transposon. Only the Tn5 transposome containing the complementary sequence will hybridize to the corresponding antibody recruitment sequence and localize at the target site. In addition to the specific complementary sequence, each Tn5 transposome also contains a unique i5 and i7 index combination (labeled as “Tn5 Index A, B or C"). After library amplification and sequencing, identification of indexes in each sequencing read makes it possible to distinguish integration within the genome that is attributed to recruitment through each individual antibody.

[0019] FIG. 7 shows an example of how the oligonucleotide sequence conjugated to the antibody can be designed to increase the number of recruitment sites. Concatenated recruitment sequences separated by spacers enable recruitment of multiple Tn5 transposomes. The recruitment nucleic acid is attached to the antibody using a click moiety.

[0020] FIG. 8 shows another example by which multiple Tn5 transposomes can be recruited to the antibody binding site. In this example, recruitment is through Tn5 transposon-transposon hybridization. Depicted are two Tn5 transposomes, one able to hybridize directly to the sequence conjugated to the primary antibody (Tn5-A), the other able to hybridize to the Tn5 transposon sequence (Tn5-B). First, Tn5-A is recruited to the target site, followed by a second incubation with Tn5-B, resulting in a secondary recruitment. The strategy could be designed in such a way as to incorporate many hybridization events resulting in the recruitment of 2, 3, 4, 5, 6, or more Tn5 transposomes to the target antibody. An additional advantage of creating a chain of TN5 molecules through successive hybridizations is that enzymatic activity is present along a distance from the antibody. Protein binding to DNA or chromatin secondary structure could prevent tagmentation near the target site and providing enzymatic activity at a greater distance from the target site may compensate by allowing tagmentation at accessible sites further from the target site.

[0021] FIG. 9 shows an example of how multiple Tn5 transposomes that are uniquely indexed can be recruited to the same antibody. In this example, multiple unique antibody recruitment sequences are conjugated to one antibody. FIG. 9A: A primary antibody is directly conjugated with two or more single stranded unique oligonucleotides each containing a unique Antibody Recruitment Sequence. FIG. 9B: A primary antibody is conjugated with one single stranded oligonucleotide that contains two or more concatenated antibody recruitment sequences that may be separated by a spacer. In both FIG. 9A and 9B, multiple recruitment sequences are conjugated to the antibody and the complement to each recruitment sequence is loaded into Tn5 as part of the transposon. In addition to the specific complementary sequence, each Tn5 transposome also contains a unique i5 and i7 index combination (labeled as “Tn5 Index A or B or C"). Each Tn5 transposome will hybridize to the corresponding antibody recruitment sequence, resulting in recruitment of multiple Tn5 transposomes to one target location. After library amplification and sequencing, identification of indexes in each sequencingread makes it possible to distinguish integration within the genome that is attributed to antibody- mediated recruitment through each individual Tn5 transposome. For simplicity conjugation of only two and three recruitment sequences is depicted but two, three, four, five, six or more can be conjugated.

[0022] FIG. 10 shows how Tn5 fusion proteins containing a protein A (pA), protein G (pG) or protein A / protein G (pAG) region can be used to recruit Tn5 transposomes through hybridization. FIG. 10A: A singleplex example is shown where pA-, pG- or pAG-Tn5 is loaded with transposons that contain a recruitment sequence. The pA-, pG- or pAG-Tn5 transposome can be recruited to the primary antibody through binding of the pA-, pG- or pAG portion of the fusion protein and the Fc region of the antibody. Tn5 transposomes that have been loaded with transposons containing the complementary sequence to the recruiting sequence can then be recruited to the target location, amplifying the amount of Tn5 transposomes. FIG. 10B: A multiplexed example is shown where two or more primary antibodies (Antibody 1 and Antibody 2) are pre-bound to pA-, pG- or pAG-Tn5 uniquely loaded with transposons containing a single stranded recruiting sequence, specific only to that complex. The pre-bound complexes will then bind the antibody target site upon incubation with the sample. Tn5 transposomes loaded with transposons containing DNA sequences complementary to a specific recruiting sequence (“hybridization sequence”) can be added. In addition to the specific complementary sequence, each Tn5 transposome also contains a unique i5 and i7 index combination. Each Tn5 transposome will hybridize to the corresponding antibody recruitment sequence and after library amplification and sequencing, identification of indexes in each sequencing read makes it possible to distinguish integration within the genome that is attributed to recruitment through each individual antibody.

[0023] FIG. 11 shows how pA-, pG- or pAG-Tn5 fusion proteins, combined with a secondary antibody can be used to increase the amount of Tn5 transposomes at the target site through hybridization. First, secondary antibodies raised against different species are pre-bound to pA-, pG-, or pAG-Tn5 transposomes that have been loaded with transposons containing a recruitment sequence. The species of the pre-bound secondary antibody complexes will correspond to different primary antibodies bound at the target site. Once the complexes are bound at the target site through primary-secondary antibody interactions, Tn5 transposomes loaded with transposons containing a sequence complementary to a unique recruiting sequence will hybridize with the corresponding sequence. In addition to the specific complementary sequence, each Tn5 transposome also contains a unique i5 and i7 index combination. After library amplification and sequencing, identification of indexes in each sequencing read makes it possible to distinguish integration within the genome that is attributed to recruitment through each individual antibody.

[0024] FIGs. 12A-12C show three combinations of recruitment and hybridization molecules in transposomes, showing that CUT&Tag libraries can be made by DNA hybridization recruitment of Tn5. 12A depicts a primary antibody bound to a target on chromatin. Antibody is bound by pA-Tn5 with a transposase loaded only with p7. The addition of a second transposome loaded with a p5 transposon that also contains a single strand region that is antisense to p7 creating a hybridization to p7 on the in the first transposome. Note: library amplification is only possible if both p7 and p5 are integrated into the genome. 12B depicts a primary antibody bound to a target on chromatin. Antibody is bound by pA-TN5 with a transposase loaded only with p7. The addition of a second transposome loaded with a p5 transposon that contains no opportunity for hybridization to the first transposome serves as a negative control. 12C depicts an antibody bound to a target on chromatin. Antibody is bound by pA-TN5 with transposase 1 loaded with transposon containing p7 and transposase 2 loaded with transposon containing p5. This is a positive control reaction and creates a library.

[0025] FIGs. 13A and 13B show gels of DNA generated from library amplification, demonstrating the ability of each of the combinations of transposomes of FIG. 12A, 12B and 12C to cleave and add amplification primer sequences to DNA. 13A: Target is the H3K4me3 (Histone H3 Lysine 4 Trimethylation) (activating mark). 13B: Target is H3K27me3 (Histone H3 Lysine 27 Trimethylation) (repressive mark). After H3K4me3 and H3K27me3 CUT&Tag reactions and amplification with p7 and p5 primer, PCR reactions were run on Tape Station. Positive control Transposomes containing p7 and p5 create a library (lane C in each Tape profile). Providing pA-TN5 with p7 transposon and separately TN5 with p5 transposon results in no library amplification (lane B in each Tape profile). Providing pA-TN5 with p7 transposon and a separate TN5 with a transposon containing p5 and a p7 hybridization site (lane A) results in amplification of a library showing that hybridization directed tagmentation is possible.

[0026] FIG. 14 shows CUT&Tag libraries made using standard protocol with primary and secondary antibody to increase recruitment of Protein A-Tn5 fusion (A,B), Standard CUT&Tag without secondary antibody (C,D), CUT&Tag libraries made by native non-protein A fusion Tn5 recruited to antibody conjugated to an oligonucleotide (E,F)SUMMARY

[0027] Methods disclosed herein overcome the challenges mentioned above by:1. Enabling reliable multiplexing and multiomic mapping through integration of unique indexes / barcodes into DNA, cDNA, RNA or DNA / RNA hybrids using a method that prevents non-specific interactions between primary antibodies and the recruited enzyme.2. Simplifying the workflow, whether multiplexing or not, through the elimination of the secondary antibody or other binding agent. Instead of employing a series of antibodies or other binding proteins to recruit enzyme or protein complexes to the nucleic acid sitesof interest, methods disclosed herein simplify the process by employing nucleic acid strands bound to antibody(ies) or protein(s) targeted to such sites to then hybridize with and recruit complementary nucleic acid strands bound to such enzyme or protein complexes.

[0028] Methods disclosed herein solve the following problems, among others:1. Through a novel primary antibody / transposome coupling strategy, methods herein enable multiplexing in a CUT&Tag-like format.2. The hybridization-based coupling strategy disclosed herein is stable and prevents the possibility of transposome / antibody swapping in multiplexed applications.3. Variations of the described method allow for the recruitment of multiple transposomes to a single antibody which will result in increased tagmentation efficiency at the targeted sites.

[0029] Four unigue aspects to the methods disclosed herein compared to the existing art include:1. They provide a means of direct recruitment of Tn5 transposomes without the need for a fusion pA-Tn5 protein (as in CUT&Tag).2. They provide a means of recruiting multiple Tn5 transposomes to the targeting antibody in the absence of a secondary antibody.3. They are highly amenable to multiplexing. By providing a specific oligonucleotide hybridization recruitment strategy, multiple Tn5 transposomes can be recruited to each specific antibody within a mix of antibodies bound to target sites.4. They are highly amenable to multiomic investigation and discovery. Any site within the genome or epigenome where an antibody can bind, Tn5 transposomes can be recruited and unigue indexes can be inserted. Epigenomic and transcriptomic targets can easily be multiplexed in a single sample.

[0030] As discussed below, there are additional embodiments of this invention wherein molecules and complexes comprising, for example, proteins and / or enzymes other than Tn5 are recruited to, or proximal to, the target nucleic acid site, which share analogous unigue aspects and problem solving compared to the existing art:DETAILED DESCRIPTIONI. Introduction

[0031] Provided herein are methods and compositions for targeting and analyzing features in nucleic acids in chromatin, in some embodiments the methods comprise: a) contacting chromatin with a recruitment complex comprising a binding agent and a recruitment nucleic acidmolecule comprising a recruitment sequence, and allowing the binding agent to bind a target in the chromatin; b) contacting the recruitment sequence with a modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence, and allowing the hybridization sequence to hybridize with the recruitment sequence; and c) allowing (e.g., by activating) the nucleic acid modification enzyme to modify nucleic acid in the chromatin. The contacting steps can be performed in either order. Subsequently, the modified nucleic acid can be analyzed, for example, by nucleic acid amplification and / or sequencing.

[0032] As used herein, the terms “bind” and “bound” refer to covalent or stable non-covalent attachment or association between two entities. This includes, for example, covalent attachment of one molecule to another, such as through an azide linkage. It also includes non-covalent binding, such as the interaction between an antibody and its target epitope, the binding between a transposase and a transposon (e.g., through a transposase recognition sequence), the binding between Protein A or Protein G and the Fc portion of an antibody, the hybridization between two complementary nucleotide sequences (e.g., of at least 8 nucleotides), and the binding between streptavidin and biotin. Stable non-covalent binding is characterized by high affinity and low dissociation rates, in contrast to transient interactions, such as enzymesubstrate binding during catalysis. As such, these terms refer to stable binding, rather than transient binding.

[0033] Chromatin is a complex of DNA and proteins found in the nuclei of eukaryotic cells. Chromatin comprises, most prominently, DNA wound around histone proteins. It can exist as euchromatin or heterochromatin. Heterochromatin is tightly compact, while euchromatin has a less compact structure. Euchromatin is typically associated with a variety of other proteins, such as enzymes, that interact with DNA. These include, for example, polymerases, transcription regulatory factors, histone modifying enzymes, chromatin remodelers, histone chaperones, methyltransferases, acetyltransferases, and transcription factors. DNA in chromatin can be modified DNA, such as methylated DNA. RNA also is associated with chromatin, typically as a result of transcription, and can be present in the form of a DNA / RNA hybrid. All of these elements can be the targets of binding agents in the methods of this disclosure.

[0034] The terms “recruitment sequence,” “hybridization sequence,” and “annealing sequence” are used herein to describe nucleotide sequences comprised in a nucleic acid molecule attached to a functional element, such as a binding agent or a modification complex, and configured to hybridize with nucleotide sequences of other nucleic acid molecules attached to other functional elements. Which of these names is used to call a nucleic acid molecule depends on context. Typically, a “recruitment sequence” is a sequence that recruits a modification complex to a binding agent. Accordingly, as discussed herein, a “recruitmentsequence” may be attached to a binding agent. In some embodiments, for example, as depicted in Figs. 8, 10 and 11 , a sequence on a modification complex is used to recruit other modification complexes. In this case, the sequence may be referred to as a “recruitment sequence.” Sequences in modification complexes that are recruited to other modification complexes may be called “hybridization sequences” or “annealing sequences,” depending on context. Accordingly, the terms are not meant to limit the particular complex to which they are attached, or where in a chain of attached complexes they are located, but are primarily used to distinguish nucleotide sequences in a larger complex comprising chains or branched modification complex structures.II. Recruitment Complexes

[0035] Recruitment complexes comprise a binding agent bound to a recruitment nucleic acid molecule comprising a recruitment sequence. The attachment can be covalent or non- covalent, as long as the recruitment molecule is stably bound to the binding agent.A. Binding Agents

[0036] Any molecule that can preferentially or specifically bind a target in chromatin can be a binding agent in the methods disclosed herein. A binding agent “preferentially binds” a target if it binds that target with at least as great, or greater, affinity than it binds any other target. A binding agent “specifically binds” a target if it binds that target with a Kd of less than 10'7M.1. Antibodies

[0037] In one embodiment, the binding agent comprises an antibody. Antibodies include fully assembled, tetrameric antibodies, such as IgG, IgA, IgE, or IgM. Antibodies also include binding fragments of antibodies or assembled portions of antibodies, such as Fab, scFV, singledomain antibody (sdAb) (aka, “nanobody”), and diabodies. Typically, binding portions include heavy and light chain variable regions containing CDRs that, together define the specificity of the antibody.2. Chromatin Binding Agents

[0038] In other embodiments, the binding agent comprises a non-antibody molecule that binds to a target as discussed herein. These include, without limitation, proteins and nucleic acids. Proteins that bind targets in chromatin include those comprising a DNA binding domain (e.g., a bZIP domain, a helix-loop-helix, a helix-turn-helix, a leucine zipper, or a zinc finger). It further includes a lexitropsin. It further includes proteins that interact with chromatin (e.g., proteins comprising an MG-box, histone methylases, recruitment proteins, Swi proteins, chromodomain or bromodomain). The binding agent also can comprise a nucleic acid molecule, such as RNA or DNA, that has specificity for a specific target (such as a target nucleotide sequence) in DNA or RNA of chromatin.B. Recruitment Nucleic Acid Molecules

[0039] The binding agent can have bound thereto a recruitment nucleic acid molecule comprising a recruitment nucleotide sequence (“recruitment sequence”). One function of the recruitment nucleotide sequence is to attract a nucleic acid modifying enzyme attached to a nucleic acid molecule having a hybridization nucleotide sequence (“hybridization sequence”) capable of hybridization with the recruitment nucleotide sequence. In certain embodiments, the recruitment nucleic acid molecule comprises DNA.

[0040] The recruitment nucleotide sequence and the hybridization nucleotide sequence have sufficient complementarity to form a stable hybrid complex. In certain embodiments, the two sequences are exactly complementary to one another. However, exact complementarity is not necessary for the formation of a stable hybrid. For example, nucleotide sequences of 15-20 bases can form stable hybrids with as little as 90% homology. Accordingly, the two sequences can have at least any of 90%, 95% or 98% homology.

[0041] In certain embodiments, the recruitment nucleotide sequence is at least any of 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides long. In other embodiments, the recruitment sequence comprises at least 10 nucleotides (e.g., at least 15 nucleotides, at least 25 nucleotides, at least 50 nucleotides, between 10 and 25 nucleotides, or between 18 and 25 nucleotides).

[0042] The recruitment sequence portion of (and the hybridization sequence portion of a hybridization molecule) is preferably single-stranded. However, the entire molecule need not be single-stranded.

[0043] Attachment of the recruitment nucleic acid to the binding agent can be covalent or noncovalent. Several methods of attaching oligonucleotides to proteins are known in the art. These include, for example, direct conjugation (e.g., click chemistry), affinity binding (e.g., biotin-streptavidin interaction), and ionic interactions (e.g., use of a positively charged moiety to attract nucleic acid). In one embodiment described in more detail herein, attachment is made using an azide linkage or click chemistry. Click chemistry is a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction that joins an azide and a terminal alkyne to form a stable 1,2,3- triazole ring. In another embodiment, nucleic acids can be attached using NHS ester-amine coupling. In another embodiment, nucleic acids can be attached using maleimide-thiol coupling. In another embodiment, nucleic acids can be attached using EDC / NHS carbodiimide coupling.

[0044] The recruitment complex can be configured to recruit one or a plurality of modification complexes. Configurations for attracting a plurality of modification complexes include attaching a plurality of recruitment nucleic acid molecules, each comprising a recruitment sequence, to the binding agent; attaching a recruitment molecule comprising a plurality of recruitment sequences to the binding agent; and combinations of these.

[0045] For example, the recruitment complex can comprise one or a plurality (e.g., at least any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20) of recruitment nucleic acid molecules. Different recruitment nucleic acid molecules can have the same or different recruitment nucleotide sequences. In the case in which recruitment nucleic acid molecules comprise more than one recruitment nucleotide sequences, combinations can include, for example, all molecules bearing the same recruitment sequences; each molecule bearing at least one recruitment sequences different than a recruitment sequence of at least one other molecule; at least one molecule bearing a recruitment sequence the same as at least one other molecule. An embodiment of this format is depicted in FIG. 3. A PEGylated antibody comprises a plurality of click moieties. Each of these can be attached to a recruitment nucleic acid molecule.

[0046] Also, the recruitment nucleic acid can comprise a plurality (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6) of recruitment nucleotide sequences. These sequences can be the same or different. In this way, the recruitment nucleic acid can recruit multiple copies of nucleic acid modification enzymes bearing the same hybridization nucleotide sequence, or nucleic acid modification enzymes bearing different hybridization nucleotide sequences Different recruitment sequences can be separated by a spacer, e.g., like bulbs on a string of lights. See, e.g., FIG. 7. Spacers can be, for example, at least at least any of 5,10, 20, or 50 nucleotides. Preferred spacers are between 5 and 20 nucleotides. Same or different hybridization sequences can be incorporated into modification complexes having either same or different modification enzymes.III. Nucleic Acid Modification Complexes

[0047] A recruitment complex associates through hybridization with a nucleic acid modification complex.

[0048] Nucleic acid modification complexes comprise a nucleic acid modification enzyme and a hybridization nucleic acid molecule comprising a hybridization nucleotide sequence.A. Nucleic Acid Modification Enzymes

[0049] The nucleic acid modification enzyme can be any enzyme that modifies nucleic acid. This includes, in particular, enzymes that cleave nucleic acids specifically or non-specifically. In one embodiment, a nucleic acid modification enzyme comprises a transposase, as described in more detail herein. However, this disclosure contemplates other embodiments, including, for example, various endonucleases such as a DNase, an MNase (which digests single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and RNA with a preference for AT-rich sequences), a DNA nicking enzyme, a biotinylating enzyme, an endonuclease (e.g., a restriction enzyme, endonuclease I), an RNAse (e.g., RNase H), a topoisomerase, a reverse transcriptase, a polyA polymerase, a terminal transferase, Cas9, Cas12, TALENs, or T7. In the case of MNases, the single stranded portion of the hybridization molecule can be protected fromdigestion by, for example, using different nucleic acid chemistries, such as phosphorothioate bonds, locked nucleic acids (LNAs) or peptide nucleic acids (PNAs).B. Hybridization Nucleic Acid Molecules

[0050] The hybridization nucleic acid molecule, as discussed above, comprises a typically single-stranded region comprising the hybridization nucleotide sequence. The hybridization nucleic acid molecule can be bound to the nucleic acid modification enzyme covalently or noncovalently, as appropriate to the application. As discussed below, when the nucleic acid modification enzyme comprises a transposase, the hybridization nucleic acid molecule associates through noncovalent binding between the transposase and a transposase recognition sequence, such as, a mosaic end sequence. In the case of other enzymes, the hybridization nucleic acid molecule can be associated through a covalent bond using linking chemistries known in the art. In certain embodiments, the hybridization nucleic acid molecule comprises DNA.

[0051] In certain embodiments, the hybridization nucleotide sequence is at least any of 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50 or 100 nucleotides long.

[0052] When a hybridization complex having a hybridization nucleotide sequence is contacted with a recruitment complex having a recruitment nucleotide sequence that can hybridize with the hybridization nucleotide sequence, the two can bind, forming a larger complex. Upon activation (if necessary), the modification enzyme modifies nucleic acids in the vicinity of the bound target.

[0053] Any complex in which a modification complex is bound, directly or indirectly, to a binding agent can be referred to as a “binding agent-modification complex”.

[0054] In one embodiment, the nucleic acid modification enzyme comprises a transposase. Transposases cleave DNA and attach transposons to the cleaved ends. Transposons can comprise various elements useful for analysis of the DNA to which they are attached. These include, for example, sequencing adapters compatible with amplification and sequencing of DNA. In another embodiment, the transposon has attached thereto a detectable label, such as a fluorescent label. After insertion into the DNA, the location of the detectable label can be detected by appropriate detection methods. For example, a fluorescent label can be detected visually, e.g., by microscopy. In multiplexed methods of this disclosure, different binding agents can be associated with transposomes comprising transposons with fluorescent labels of different colors. Transposases will not release fragmented DNA from chromatin until they are removed by, e.g., proteases (e.g., proteinase K) or a detergent (e.g., SDS). Accordingly, the geographical location of the binding in the nucleus can be determined.IV. Recruitment of Multiple Modification Complexes Using Other Modification Complexes Functioning as Recruitment Molecules

[0055] Another embodiment of the method uses modification complexes comprising nucleic acids comprising first nucleotide sequences to recruit (“recruitment sequence”) other modification complexes comprising nucleic acids having second sequences to which the first sequences can hybridize (“hybridization sequence” or “annealing sequence”).

[0056] In certain embodiments, a modification complex is attached, directly or indirectly, to a binding agent. In other embodiments, a modification complex is attached in a linear or branched structure to other modification complexes.A. Multiple Modification Complexes in Linear Chain and Branched Structures

[0057] By manipulating the number and combination of hybridization sequences in a modification complex, tandem or branching structures comprising a plurality of modification complexes can be formed. The number of modification complexes ultimately bound to a single binding agent can be at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. By recruiting more modification enzymes to sites of the binding agent, binding modification reactions can become more efficient.

[0058] For example, any modification complex provided with each of a hybridization and recruitment sequence can recruit one or a plurality of other modification complexes in at least a chain arrangement, e.g., like bulbs on a string. If a modification complex is provided with two, three, or more hybridization sequences, then, depending on the configuration, branched structures of modification complexes can form, e.g., like a tree decorated with lights. See, e.g., FIGs. 8, 9, 10 and 11. Accordingly, the same nucleotide sequence can function in different modification complexes as a “recruitment sequence” to recruit another modification complex, or as a “hybridization sequence” or “annealing sequence” to bind a modification complex to another modification complex.B. Binding Of Modification Complex To Recruitment Complex Through Hybridization Sequence On Recruitment Complex

[0059] One exemplary embodiment is shown in FIG. 8. There, a first modification complex (transposome) comprises a hybridization sequence designed to hybridize with a recruitment sequence on a binding agent. The first modification complex further comprises a recruitment sequence designed to recruit a second modification complex (another transposome) through a “hybridization sequence” or “annealing sequence” attached to the second modification complex. In this embodiment, the second modification complex can further comprise another recruitment sequence which, in turn, can recruit another modification complex through a hybridization or annealing sequence. These modification complexes, in turn, further comprise other recruitmentsequences to which hybridization or annealing sequences on other modification complexes can hybridize. In this way, chains comprising a plurality of modification complexes can be formed.C. Binding Of Modification Complex To Binding Agent Through An Affinity Agent

[0060] In another embodiment, a modification complex comprising a recruitment sequence binds to a binding agent through an affinity agent. As used herein, an “affinity agent” is a molecule or complex that has affinity for a binding agent. Binding can be direct or indirect.

[0061] An example of direct binding is the use of a single molecule (e.g., protein A or protein G) bound to the modification complex and binding directly to the binding agent.

[0062] An example of indirect binding is the use of an affinity agent comprising a plurality of affinity elements bound together and including a “terminal affinity element” and one or more “intermediate affinity elements”. This affinity agent is bound to the modification complex and to the binding agent. An example of this is protein A or protein G (the intermediate affinity element), attached to an antibody with affinity for the binding protein (the terminal affinity element). The affinity agent can be bound to the modification complex through the nucleic acid modification enzyme or the nucleic acid moiety. Attachment between the affinity agent and the modification complex can be covalent or noncovalent. For example, the affinity agent can be comprised in a fusion protein with the modification enzyme.1. Direct Binding

[0063] An example of direct binding is shown in FIG. 10. A binding agent-modification complex is produced by contacting a binding agent (in this case, an antibody) with a modification complex (in this case, a transposome) that comprises a nucleic acid modification enzyme (in this case, a transposase), a nucleic acid molecule comprising a recruitment nucleotide sequence (in this case a transposon comprising a transposase recognition sequence and a “recruitment sequence”), and an affinity agent (in this case, protein A or protein G) that has affinity for the binding agent (antibody). In some embodiments, the affinity agent and the nucleic acid modification enzyme can be combined in a fusion protein, for example (as in this case), a transposase-protein A fusion protein. This complex can be formed before or after the binding agent is bound to its target in the chromatin. However, as discussed below, formation of a binding agent-modification complex before contact with chromatin allows for multiplexing.(See Fig. 10B.)2. Indirect Binding

[0064] In another embodiment, the affinity agent comprises a chain comprising a terminal affinity element having affinity for the binding agent, and one or more intermediate affinity elements that link the terminal affinity element with the modification complex. An affinity agentcan comprise, for example, at least any of one, two, three, or four intermediate affinity elements. An example of this embodiment is depicted in FIG. 11. Here, the binding agent comprises a Primary Antibody. The modification complex comprises modification enzyme (in this case, a transposome) comprising a recruitment nucleic acid molecule comprising a recruitment sequence (here, a transposon comprising a transposase recognition sequence and a recruitment sequence). The modification complex is attached to a complex affinity agent that comprises a terminal affinity element (in this case, a secondary antibody) attached to an intermediate affinity element (in this case, Protein A or Protein G). The affinity agent is bound to the modification complex (here, as a fusion protein between the transposase and protein A / protein G). Again, as discussed below, such constructs are useful in multiplexing methods, in which different terminal affinity elements bind different binding agents.V. Targeted Nucleic Acid ModificationA. Target Binding

[0065] The methods disclosed herein embrace embodiments in which the binding molecule is attached to a recruitment molecule in a recruitment complex, e.g., as in FIG. 4, FIG. 6, FIG. 7, FIG. 8, and FIG. 9, as well as those in which the binding molecule is attached to the modification complex through an affinity agent on the modification complex, e.g., as in FIG. 10, and FIG. 11.

[0066] In certain embodiments of this disclosure, a binding agent is contacted with chromatin, and allowed to bind to its target in the chromatin. The composition to be contacted can be whole cells, permeabilized cells, nuclei, permeabilized nuclei, chromatin, isolated chromatin, or fragmented chromatin, a support, such as a glass slide, having a surface supporting a tissue slice, all of which may be native or fixed.

[0067] Targets in chromatin include, without limitation, a histone, a histone comprising a post-translational modification (e.g., by methylation, acylation, phosphorylation, or ubiquination), a chromatin-associated protein (e.g., a chromatin remodeling protein or a polycomb group (PcG) protein), a topologically associating domain (TAD), a chromatin loop, a modified RNA, a modified DNA (e.g., methylated DNA), a cDNA, a DNA / RNA hybrid (e.g., R-loops), a transcription factor, a single stranded DNA binding protein, a DNA repair associated protein, a transcriptional machinery protein, and a DNA replication associated protein. Any modification of any histone can be a target, e.g., modifications to any of H1, H2A, H2B, H3, and H4. Specific, non-limiting examples include, H1 acetylation or phosphorylation, H2A.X phosphorylation, H2A.Z acetylation / methylation, H2A ubiquitination, H2B ubiquitination, H2B acetylation, H3K4me3, H3K27me3, H3K27ac, H3K4me1, H3K4me2, H3K9me2, H3K9me3, H3K9ac, H3K36me2, H3K36me3, H3K56ac, H3K79me3, H3K79me3, H4K16ac, and H4K20me3.

[0068] In some embodiments, e g., as in FIG. 4, FIG. 6, FIG. 7, FIG. 8, and FIG. 9, the nucleic acid modification complex can be contacted with the recruitment complex either before or after the binding agent binds to its target in chromatin. For example, the recruitment complex can be contacted with the chromatin and allowed to bind, and, subsequently, the nucleic acid modification complex can be contacted with the bound recruitment complex. Alternatively, before binding to chromatin, the recruitment complex can be contacted with the modification complex and allowed to associate through hybridization. This total complex can then be contacted with chromatin and allowed to bind to the target.

[0069] In other embodiments, e.g., as in FIG. 10 and FIG. 11 , the nucleic acid modification complex comprising an affinity agent can be contacted with the binding agent either before or after the binding agent binds to its target in chromatin. For example, the binding agent can be contacted with the chromatin and allowed to bind, and, subsequently, the nucleic acid modification complex can be contacted with the bound binding agent. Alternatively, before binding to chromatin, the binding agent can be contacted with the modification complex and allowed to associate through hybridization. This total complex can then be contacted with chromatin and allowed to bind to the target.B. Enzyme Catalyzation

[0070] After a larger complex comprising the DNA modification complex, the binding agent, and bound chromatin are assembled, the nucleic acid modification enzyme is allowed to catalyze the modification of nucleic acid. Catalyzing can occur spontaneously based on cofactors in the mixture, or can involve activating the enzyme with a metal ion or co-factor.

[0071] Certain enzymes, in particular endonucleases, can require the presence of metal ions to be active. Such ions can include, for example, Mg++, Ca++, Mn++Zn++, Co++, and Ni++. In other cases, the enzyme may be activated by a co-factor such as ATP or NADPH.

[0072] Before activation, the mixture can be washed to remove unbound complexes. Also, washing can remove cofactors that could activate enzymes before binding. Such factors, can be added back in as part of the activation step. In the case of nuclei, these factors are washed out after permeabilization of the nuclei. Alternatively, they can be neutralized, for example, with chelators such as EDTA or EGTA. In order to activate the enzyme, co-factors, such as Mg++or Ca++can be added in excess.

[0073] Upon activation, the DNA modification enzyme will perform its particular modification operation on nucleic acid in the chromatin. Depending on the enzyme, this could involve nicking or cleaving DNA, RNA or a DNA / RNA hybrid, or inserting an adaptor molecule to one or both ends of a cleaved nucleic acid molecule. For example, if the nucleic acid modification enzyme comprises a restriction endonuclease, the product will be a nucleic acid fragment comprising a 5’ overhang in the form of a “sticky end.” Such adaptors can be provided with moieties, such afluorescent molecule, or biotin, that tag the modified DNA with these moieties. Any known fluorescent labels can be used. These include, without limitation, ATTO 590 Cy5, Cy3, Alexa488, Alexa405, Alexa555, Alexa568, Alexa 647,Atto 550, Fluorescein family dyes (e.g., dichlorofluorescein, difluorofluorescein, tetrtachlorofluorescein, and hexachlorofluorescein), and Chromeo 494, as well as analogs / equivalents known in the art. The moieties can be used in separate operations to for analysis or isolation of the tagged DNA. A GC or adenine methyltransferase will methylate at bases in proximity to the target site. Sequencing (most likely on a platform that can recognize these modifications, i.e. , Oxford Nanopore or PacBio can identify the proximity of the target site. After nicking the nick can be filled in with biotinylated nucleotides. Then after DNA fragmentation regions proximal to the target site can be enriched by streptavidin.

[0074] Because the nucleic acid modification complex is bound to the binding agent, it will perform its work on a site in the chromatin proximal to the binding location. When the action involves cleavage of nucleic acid, enzymes may cleave nucleic acid on either side of the binding agent binding site, producing a nucleic acid fragment in which the binding site is between the two ends of the nucleic acid molecule. In this way, the binding site can be mapped to the genome.VI. Nucleic Acid Analysis

[0075] After nucleic acid modification, the modified nucleic acid can be analyzed. Analysis can be performed with or without prior isolation of the modified nucleic acid.

[0076] Nucleic acid analysis can include, but does not require, isolation of the modified nucleic acid. This can involve dissociating the nucleic acid from the associated proteins and performing specific or non-specific capture of the nucleic acids. Dissociation can include treatment with proteases, such as proteinase K, to digest histone proteins and nucleic acid modification proteins, such as transposases. Non-specific capture could include, for example, isolating all nucleic acid fragment in a certain size range. This can be done, for example, by Ampure™ or SPRI bead clean-up, density gradient centrifugation, chromatography, or electrophoresis. Specific capture can include, for example, hybridization of probes, e.g., bound to solid supports, to specific sequences in the nucleic acids, such as specific genomic sequences or sequences contained in nucleic acid adapters attached to the ends of cleaved DNA.

[0077] In one embodiment, analysis comprises DNA amplification. DNA amplification can comprise any known method of amplification. This includes, without limitation, PCR, rolling circle, Loop- Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Strand Displacement Amplification (SDA) Multiple Displacement Amplification (MDA), Nucleic Acid Sequence-Based Amplification (NASBA), Recombinase Polymerase Amplification(RPA), and Hybridization Chain Reaction (HCR). When DNA modification comprises adding DNA sequences adapted for an amplification reaction, amplification can be performed in situ without further isolation of the modified DNA. For example, PCR, LAMP, transcription-mediated amplification, strand displacement amplification, and recombinase polymerase amplification require primer binding sequences.

[0078] Analysis can include DNA sequencing and analysis of the resulting sequence reads. DNA sequencing can include high-throughput sequencing. High-throughput sequencing methods include, for example, pyrosequencing, sequencing-by-synthesis (e.g., Illumina sequencing, Ion Torrent sequencing, PacBio (SMRT sequencing)), sequencing by ligation (e.g., SOLiD sequencing, nanoball sequencing (Complete Genomics)), nanopore sequencing (e.g., Oxford Nanopore Technologies), sequencing-by-hybridization (e.g., DNA microarrays and sequencing on beads), synthetic long read sequencing (e.g., linked-read sequencing of 10X Genomics), single molecule sequencing (e.g., Helicos), and pyrosequencing (e.g., Roche 454 sequencing). Such method can use platform-based adaptors. Accordingly, in the nucleic acid modification reactions of this disclosure, such adaptors can be incorporated into the product (for example, by ligation during transposition). It is understood that sequencing DNA fragments produced the methods herein embraces sequencing the molecule, itself, or sequencing amplified product of the molecule.

[0079] Analysis can also include methylation mapping, that is, determination of methylation sites in the DNA. Methylation mapping typically involves conversion of one class of bases, for example cytosine or 5-methyl cytosine, into another form, such as uracil. Upon sequencing, methylated bases can be detected based on the presence or absence of a modified or nonmodified base at a position. For example, if cytosine, but not modified cytosine, is converted to uracil, then a read of “C” at the position indicates this base had been modified, while a read of “U” indicates non-modified cytosine. Methylation can also be detected by direct sequencing (i.e. , without amplification) on Oxford nanopore or PacBIO single-molecule sequencing systems.VII. Multiplex Analysis

[0080] Methods described herein can be multiplexed to target more than one different target in chromatin in a single experiment. Multiplex methods contemplate at least any of 2, 3, 4, 5, 6, 7, 8, 9 or 10 different binding agents.A. Direct Recruitment

[0081] In one embodiment of multiplexing, modification complexes are directly recruited to recruitment complexes through binding between a recruitment sequence on the recruitment complex and a hybridization sequence in the modification complex. In this embodiment, exemplified in FIG. 6, a plurality of different recruitment complexes comprising binding agents having affinity for different targets have different recruitment sequences. These recruitmentcomplexes are contacted with chromatin and allowed to bind. A plurality of different modification complexes comprising different hybridization sequences are contacted with the recruitment complexes. The different hybridization sequences hybridize with their counterpart recruitment sequences on the recruitment complexes. In this embodiment the different modification complexes can be provided with molecular identifiers, e.g., unique molecular identifiers, that indicate the specific binding agent to which the modification complex is attracted. After modification, the modified DNA is tagged with the identifying sequence. Upon sequencing, sequence reads can be deconvoluted to determine which different binding agent and, therefore, which different target, is proximal to the modification site.

[0082] In FIG. 6, each binding agent comprises an antibody specific to a different protein in the chromatin. The modification complexes comprise transposomes comprising different hybridization sequences. The different hybridization sequences pair with different recruitment sequences. Different transposons can further comprise unique index combinations. After modification and sequencing, sequence reads can be demultiplexed.B. Binding Agent-Modification Complex

[0083] In another embodiment of multiplexing, a binding agent-modification complex (as discussed above) is formed that comprises a binding agent bound to a modification complex through an affinity agent having affinity for the binding agent. This embodiment is exemplified in FIG.10B. In this embodiment, a modification complex comprising a nucleic acid modification enzyme bound to a nucleic acid molecule comprising a recruitment sequence is provided. The modification complex further comprises attached thereto, an affinity agent. This complex is contacted with a binding agent to form a stable binding agent-modification complex. In the embodiment exemplified in FIG. 10B, the binding agent comprises an antibody. The modification complex comprises a transposome comprising transposases bound to transposons comprising transposase recognition sequences and an overhang comprising a recruitment sequence. The modification complex further comprises an affinity agent, in this case, a protein A or protein G moiety which, in this case, is attached to a transposase in the form of a fusion protein. In this case, the affinity agent binds the binding agent through specific affinity for the Fc portion of the antibody.

[0084] A plurality of such binding agent-modification complexes are provided to include a plurality of different binding agents that each bind to different targets. These complexes are contacted with chromatin and allowed to bind to their targets. Because the modification complexes comprise a recruitment sequence, they can be used to attract other modification complexes that comprise hybridization or annealing sequences that hybridize to the recruitment sequences. In this way, multiple modification complexes can be recruited to the binding sites. Furthermore, if the nucleic acids in the modification complexes are provided with molecular identifiers that correspond to the binding agent which they are bound, the sequences can beused to determine which binding agent attracted the modification complex to a particular locus in the genome.

[0085] In this embodiment, if the affinity agent does not distinguish between different binding agents, preparation of binding agent-modification complexes is performed in advance of contacting binding agents with their targets in chromatin. In this way, also, the collection of binding agent-modification complexes can be mixed, and the mixture can be contacted with the chromatin.C. Affinity Agent-Modification Complex

[0086] In another embodiment, a modification complex is recruited to particular binding agent through an affinity agent having specific affinity for the binding agent. That is, the modification complex is attached to such an affinity agent. This embodiment contemplates both direct attachment of the affinity agent to the modification complex, and indirect attachment, that is, through an affinity agent comprising a terminal affinity element having specificity for a particular binding agent or class of binding agents, and one or more intermediate affinity elements that bind the terminal affinity element to the modification complex. Complexes comprising a modification complex bound to an affinity agent can be referred to as an “affinity complex.”

[0087] In one embodiment, the affinity complex comprises a complex affinity complex that comprises a terminal affinity element and one or more intermediate affinity elements. This construct is described in more detail above. In any case, a plurality of different affinity complexes are provided. Each affinity complex comprises an affinity agent that preferentially or specifically binds a different binding agent. This combination is contacted with binding agents already bound to chromatin. Each affinity agent binds to the binding agent it recognizes, thereby attaching the modification complex to the binding agent.

[0088] An exemplary embodiment of this method is shown in FIG. 11. A plurality of different binding agents (e.g., antibodies) are allowed to bind to their respective targets in the chromatin. Different affinity agent-modification complexes are provided comprising different affinity agents (e.g., species-specific antibodies) having affinity for the different binding agents. Each affinity agent-modification complex comprises a modification complex (e.g., a transposome) comprising a nucleic acid modification enzyme (e.g., a transposase) bound to a recruitment nucleic acid molecule (e.g., a transposon with transposase recognition sequences and a requirement sequence). Upon contact, the different affinity agent-modification complexes bind to their respective binding agent targets in the chromatin. Because they comprise recruitment sequences, modification complexes can recruit other modification complexes comprising annealing sequences that hybridize with their respective recruitment sequences. Again, byproviding different recruitment complexes with different molecular identifiers, upon sequencing, particular binding agents can be associated with their binding locations in the chromatin.

[0089] It is further understood that each of these multiplexing strategies can be combined with embodiments in which multiple modification enzymes are recruited to the same binding agent.VIII. Tagmentation Embodiment

[0090] Transposomes are complexes comprising a pair of transposases, each transposase associated (e.g., “loaded”) with a transposon. Transposons are DNA molecules comprising a nucleotide sequence, typically double stranded, recognized by a transposase. Transposons can further comprise regulatory elements such as promoters, enhancers, silencers and insulators. The transposases are dimerized to form a stable transposome complex. The transposases can be activated by addition of an appropriate metal ion, e.g., Mg++. FIG. 2 depicts an exemplary transposome.

[0091] Transposases catalyze a reaction in which a segment of DNA is cleaved and each end is bound to the end of a transposase recognition sequence in a transposon. Any transposase is useful in the methods of this disclosure. These include for example, Tn5, sleeping beauty, Tn7, Tn3, Tn10, Vibrio harveyi, PiggBac, Hermes, and Tol2.

[0092] Transposons useful in the methods of this disclosure comprise a double stranded portion comprising a transposase recognition sequence, and a 5’ overhang comprising a hybridization sequence (or a recruitment sequence as the case may be). The recognition sequence can be a terminal inverted repeat, which can be palindromic. Mosaic end transposons are recognized by, e.g., Tn5. One example of a mosaic end sequence is shown in FIG. 2. The recognition sequence also can comprise a synthetic sequence.

[0093] In certain embodiments, a transposase is loaded with two different transposons. One or both of the transposons can comprise a hybridization nucleotide sequence.Furthermore, the transposons can be provided with sequences useful in nucleic acid amplification. Accordingly, each end of a DNA fragment that is cut from the genome in a transposition process can be attached to different transposons. In this way, the fragment can comprise all sequences necessary for amplification or capture.

[0094] Nucleotide sequences that can be included in a transposon include, for example, those shown in FIG. 4. One sequence shown here are P5 and P7 Illumina amplification adapter sequences for binding to a flow cell. The transposon also includes a molecular index sequence. These sequences allow identification of the original molecule from a sequence read is derived. Molecular indices can be unique, that is a different sequence for each transposon, or nonunique, relying on a portion of the genomic DNA to provide unique identification. Another sequence can be a sample index sequence, which identifies the sample from which a readcame in a multiplex sequencing run. Another sequence that can be included is a priming sequence for use on a sequencing platform. Another sequence can be a T7 promoter sequence for signal amplification through in vitro transcription. In FIG. 4 these are given as Read 1 and Read 2.

[0095] This disclosure provides, in one embodiment, a method for recruiting Tn5 transposomes comprising single stranded DNA through hybridization to specific genomic locations or to target-bound antibodies conjugated to complementary single stranded DNA. Hybridization mediated targeting of the Tn5 transposome results in directed transposon integration into the genome at the targeted location. This method is described using Tn5 transposomes as the recruited element and an antibody as the recruiting element, but those skilled in the art would recognize that the recruited element could be any protein, enzyme or molecule capable of modifying the target site in a detectable way, and that the recruiting element could be any protein, enzyme or molecule with an affinity to a genomic location, genomic structure, DNA modification or, RNA, RNA structure or RNA modification, various molecules and complexes associated with or bound to these nucleic acids and structures or epigenomic epitope.

[0096] This method takes advantage of the partially single stranded DNA molecules that are used to load Tn5, and the predictable and specific nature of hybridization between two complementary single stranded DNA molecules, or other analogs which hybridize. In one embodiment, Tn5 dimers (referred to as “Tn5 transposomes”) are the active form of the transposase protein and require two Tn5 monomers to be mixed with two partially double stranded DNA molecules containing a specific mosaic end sequence and a 5’ single strand overhang (ME; 5’-NNNNNNNNNNNNNNNNCTGTCTCTTATACACATCT-3’, where is A,T, G, C) (SEQ ID NO:1), referred to as a transposon, in a process referred to as “loading” (FIG. 2). During loading, each Tn5 monomer binds the ME sequence and subsequently dimerizes. The sequence used to load Tn5 must contain the double stranded ME sequence but can also contain a single stranded 5’ overhang. There are no specific sequence requirements for the overhang and thus it is a customizable region. In the process of tagmentation, DNA cleavage occurs and the ME sequence and the single strand overhang are added onto the cut end of the genomic DNA, RNA, RNA / DNA hybrid or chromatin, enabling a method of inserting customizable sequences into regions of interest. In addition the transposon and transposome may comprise other elements that are useful for detection and mapping of genomic and epigenetic features, for example bar codes and other identifying sequences or molecules, priming adaptors as discussed below, etc.

[0097] Once Tn5 is bound to the transposon (loaded), the double stranded portion of the transposon is within the active site of the enzyme, while the single stranded overhang protrudes (FIG. 2). With this knowledge, methods disclosed herein use the customizable protruding singlestranded overhang to recruit the Tn5 transposome directly to a complimentary single stranded target site (FIG. 4A). In one embodiment, a unique single stranded sequence will be covalently attached to an antibody of interest (herein called “antibody recruitment sequence”) and Tn5 will be loaded with transposons wherein the overhang sequence contains a sequence that is complementary to the antibody recruitment sequence (herein called “transposome hybridization sequence”). Since the antibody recruitment sequence is unique, it will only recruit Tn5 transposomes containing the complementary overhang sequence. In addition to the complementary antibody recruitment sequence, the overhang region of the transposon can contain a multitude of different functional sequences. Here, one version of customized transposons designed for multiplexing and next generation sequencing readout is described. The transposon can contain a unique identifying index sequence, a unique molecular identifying (UM I) sequence and sequences necessary for sequencing on next generation sequencing platforms (flow cell binding and read primer binding sequences) (FIG. 4B).

[0098] The single-stranded oligonucleotide conjugated antibody will be bound to a target within the genome of a sample which may be whole cells, permeabilized cells, nuclei, permeabilized nuclei, chromatin, isolated chromatin, or fragmented chromatin, all of which may be native or fixed, thus recruiting the Tn5 transposome to that location through hybridization. Once recruited, Tn5 transposomes will be activated using a cofactor such as magnesium or cobalt to induce tagmentation. Insertion of the transposons will occur at locations proximal to the target site. Polymerase chain reaction (PCR) amplification can be used to amplify the tagmented sequences, e.g., using two different primers. The primers are designed to produce a completely double stranded fragment containing the genomic region of interest flanked by flowcell binding, read sequences and two unique indexes necessary for multiplexed next generation sequencing, and a UMI within one of the flanking regions (FIG. 5). The transposon sequences will be designed such that the unique i5 and i7 indexes will be associated with a single unique antibody recruitment complement sequence, enabling multiple antibody targeted proteins to be interrogated within the same reaction (FIG. 6). Tagmentation products generated at the target site will contain sequences (including unique indexes) inserted by the recruited Tn5 enabling bioinformatic deconvolution of the associated antibody using the Tn5 specific indexes.

[0099] The customizability of the transposon overhang enables tailoring of the functional sequence for complex applications, such as single-cell or spatial genomics mapping. Additional indexes and UMIs can be added to the transposon to yield a sequence with cell specific indexes. Alternative amplification motifs like a T7 sequence, other RNA polymerase initiation sequences, primer binding sites, or sequences required for circulation or rolling circle amplification may also be included. Additionally, each transposon may be modified with a unique fluorescent molecule to enable multiplexed detection using microscopic methods.[000100] The antibody / oligonucleotide conjugation strategy utilized can covalently attach, e.g., up to 6 oligonucleotides, encoding antibody recruitment sequences, to the primary antibody, creating as many as 6 recruitment locations for Tn5 transposomes, with an average of 6 binding sites (FIG. 3A and 3B). However, the methods by which DNA oligonucleotides or other hybridization means are attached to the antibody are numerous and any such conjugation strategy is applicable to this method. Conjugation oligonucleotides may vary in nature with respect to oligonucleotide sequence design such that the antibody recruitment oligonucleotide sequence may be a concatemer of the hybridization sequence with or without spacers in between, allowing multiple hybridization events to occur at the same antibody resulting in recruitment of multiple Tn5 transposomes (FIG. 7).[000101] The general steps of this embodiment are:1. Covalently conjugate the primary antibody / antibodies to single strand oligonucleotides that encode the recruitment sequence / sequences.2. Load Tn5 with transposons containing a single stranded region that is complimentary to the antibody recruitment oligonucleotide.3. Incubate sample with DNA conjugated primary antibody / antibodies that bind transcription factors(TFs), histone modifying enzymes, histone post translational modifications (PTMs), RNA modification, cDNA, DNA modification or any other target an antibody can bind.4. Wash away unbound primary antibody.5. Incubate the sample with Tn5 transposomes loaded with complementary antibody recruitment sequence to allow hybridization to complimentary sequences on the antibody.6. Wash away unbound Tn5 transposomes.7. Add the appropriate cofactor to activate the transposase allowing for tagmentation at the targeted sites.8. Purify and / or amplify the tagmented DNA. (Note: Amplification can proceed in situ without the need for prior nucleic acid purification.)9. Sequence the DNA and bioinformatically analyze.[000102] In an alternative approach, the Tn5 transposomes with transposons containing a single stranded region can first form a stable association with oligonucleotide conjugated primary antibodies in solution, prior to incubation with sample.1. Covalently conjugate the primary antibody / antibodies to single strand oligonucleotides that encode the recruitment sequence / sequences.2. Load Tn5 with transposons containing a single stranded region that is complimentary to the antibody recruitment oligonucleotide.3. Incubate the DNA conjugated antibody with the matching Tn5 with transposons containing a single stranded region so that, though hybridization, the antibody and barcoded transposomes are stably bound.4. Incubate the sample with the stably attached antibody / transposase complex or complexes.5. Wash away unbound complexes.6. Add the appropriate cofactor to activate the transposase allowing for tagmentation at the targeted sites.7. Purify and / or amplify the tagmented DNA.8. Sequence and bioinformatically analyze.A. Antibody Conjugation Chemistry[000103] One antibody conjugation strategy combines site specific labeling of Active Motif’s Abflex® recombinant antibodies via sortase recognition sequence and click chemistry. The combination of the two techniques enables rapid and simple site-specific conjugation of custom oligonucleotides to any Abflex® recombinant antibody. Each Abflex® recombinant antibody contains a sortase recognition sequence (LPXTG) within both heavy chains. Upon addition of sortase and a poly-glycine label, the label will be covalently conjugated to the terminal end of the antibody, leaving the antigen binding site free for target recognition.[000104] This example attaches a click chemistry moiety with the poly-glycine sortase attachment site to the antibody enabling click chemistry labeling with a custom oligonucleotide for Tn5 transposome recruitment (FIG. 3).[000105] Click chemistry is a novel strategy for selective conjugation of biomolecules in vitro or in vivo that utilizes two moieties, azide and alkyne, that do not occur in nature, to guarantee selective reactivity. The linker moiety contains either an alkyne (e.g. DBCO) or azide group that when reacted with the alternative click moiety on a custom oligonucleotide sequence forms a covalent bond, resulting in a primary antibody covalently labeled with a Tn5 transposome recruiting oligonucleotide sequence.[000106] Alternate antibody / oligonucleotide conjugation strategies would also be suitable for the described method.[000107] Steps:1. Mix Abflex® recombinant antibody, sortase A5 enzyme and (Gly)5-Click label and incubate at 30°C for one hour with shaking.2. Remove excess label using column purification.3. Inactivate sortase.4. Mix the click moiety labeled antibody with the complement click moiety labeled oligonucleotide, incubate at 4°C for 24 hours.5. Remove excess label by size exclusion or purification.B. Tn5 loading[000108] Tn5 loading requires two DNA adapters (comprised in the transposons) per Tn5 transposome. The transposons typically contain the ME sequence where a complementary sequence is annealed, making the transposon double stranded at this region. The transposon may contain additional single strand sequences which comprise the 5’ overhang (i.e. , transposon A and transposon B). In this design, the overhang of one transposon contains the complementary antibody / oligonucleotide sequence, a unique index, a unique molecular identifier and the read 1 primer necessary for sequencing on a next generation sequencing platform. The overhang of the other transposon contains the flow cell binding sequence for next generation sequencing, another unique index, and the read two sequence necessary for next generation sequencing (FIG. 4B).Steps:1. Separately, anneal Tn5 transposon A and transposon B with ME complementary sequence to make a partially double stranded transposon.2. Mix T n5 monomer with two partially double stranded transposons at a 1 : 1 molar ratio and incubate at room temperature for one hour.C. Tn5 recruitment through DNA hybridization and targeted tagmentation Steps:1. Incubate the DNA conjugated primary antibody with sample.2. Wash in 150 mM NaCI wash buffer to remove excess primary antibody.3. Incubate sample with loaded Tn5 to allow for hybridization.4. Wash in 300 mM NaCI wash buffer to remove excess, unhybridized Tn5.5. Add 10 mM MgCh to activate Tn5 transposome, perform tagmentation reaction at 37°C for 30 minutes.D. Purification and amplification of tagmented DNASteps:1. Add sodium dodecyl sulfate, proteinase K and heat at 55°C for 10 minutes to stop the tagmentation reaction and release the TN5 from the DNA.2. Purify tagmented DNA using columns or beads.3. Perform library amplification according to the table below and thermocycler program belowE. PCR conditions a) 72°C for 5 minutes b) 98°C for 30 seconds c) 10-14 cycles of: a. 98°C for 10 seconds b. 63°C for 10 seconds d) 72°C for 1 minute e) Hold at 10°CF. Sequencing and Bioinformatic Analysis[000109] Next generation sequencing on an Illumina platform can be performed using paired- end sequencing with the following parameters:Read 1 : 38 base reads Read 2: 38 base reads Index 1: 8 base reads Index 2: 8 base reads[000110] The described method can be designed to be compatible with other next-generation sequencing (NGS) platforms.[000111] The above embodiments are not exhaustive. Those of skill in the art will recognize that the hybridization based targeted recruitment technique of the method can be readily adapted for a number of applications with respect to RNA as well as DNA, chromatin etc., and can be used with a variety of known mapping, detection and other genomic and epigenetic strategies, using other enzymes, proteins, and targeting agents and for recruiting a variety of molecules and complexes, in addition to Tn5 and transposomes, to nucleic acid(s) and their associated molecules for such applications. Some exemplary, but not exhaustive, additional embodiments are described below.IX. Kits[000112] Further provided herein are kits comprising reagents and devices for performing methods as disclosed herein. Elements of a kit, such as reagents, can be comprised in a container, such as a stoppered tube, a box, or an envelope.[000113] Kits of this disclosure comprise at least a set of one or more binding agents, such as antibodies, that target one or more targets in chromatin. The binding agents optionally can be configured as recruitment complexes. Alternatively, the components of a recruitment complex,such as a binding agent and a recruitment nucleic acid molecule, can be contained in separate containers, for assembly by the user.. The different targets may fall into the same classes, such as different histone modifications, different histone modifying enzymes, or different transcription factors. So, for example, a kit could comprise at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 500 or 1000 containers, each container containing a binding agent that binds to a different target.[000114] Kits of this disclosure also can comprise at least a set of modification complexes. These can be configured as assembled complexes, or separately as modification enzymes, such as transposomes, and recruitment nucleic acids, such as transposons. In some embodiments, there can be a plurality of containers, each container containing a different modification complex comprising a hybridization sequence that hybridizes to a recruitment sequence in the kit. In another embodiment, a plurality of different modification complexes can be contained in a single container.[000115] Accordingly, in one embodiment, a kit can comprise at least (a) at least one container containing a recruitment complex (or containers containing its components), , such as an antibody attached to a hybridization molecule, and (b) at least one container containing a nucleic acid modification complex (or containers containing its components), such as a transposome loaded with a hybridization molecule (which also can be separately contained in different containers).[000116] Kits also can comprise one or more solutions useful in performing the methods describe herein.[000117] Accordingly, in addition to a container containing a recruitment complex (or containers containing its components), and a container containing a modification complex (or containers containing its components), a kit can comprise items selected from:(I) a container containing a nucleic acid modification enzyme activating agent;(II) a container containing a buffer comprising a detergent (e.g., digitonin);(III) a container containing concavalin A beads (e.g., to capture cells or nuclei through binding to complex carbohydrates);(IV) a container containing a binding buffer;(V) a container containing a wash buffer;(VI) a container containing antibody buffer;(VII) a container containing protease inhibitor;(VIII) a container containing stop buffer (e.g., containing EDTA to quench the ions);(IX) a container containing Proteinase K (e.g., to digest modification enzymes, such as Tn5);(X) a container containing a solid phase for DNA capture (e.g., column or beads);(XI) a container containing dNTPs, amplification primers, and polymerase;(XII) a container containing SPRI or other beads for purification of DNA after PCR; and(XIII) a container containing a reagent for coupling a nucleic acid molecule to a protein.EXAMPLESI. Transposon-transposon hybridization recruitment[000118] The customizability of the overhang in the T n5 transposon sequence enables an additional method of recruitment through Tn5 transposon-transposon hybridization (FIG. 8). Tn5 can be loaded such that transposon A contains a sequence that is complementary to the antibody recruitment sequence, and transposon B contains an alternative sequence herein called the Tn5 recruitment sequence. An additional Tn5 transposome can be loaded with sequences that contain a complementary sequence to the Tn5 recruitment sequence, thus hybridizing directly to a recruited Tn5 transposome containing the antibody recruitment sequence. Repeated iterations of this recruitment technique using alternative sequences result in additional Tn5 transposomes directed to the target site.Steps:1. Incubate the DNA conjugated primary antibody with the sample.2. Wash in 150 mM NaCI wash buffer to remove excess primary antibody.3. Incubate sample with loaded Tn5-A.4. Wash in 300 mM NaCI wash buffer to remove excess Tn5.5. Incubate sample with loaded Tn5-B.6. Wash in 300 mM NaCI wash buffer to remove excess Tn5.7. Add 10 mM MgCh to activate Tn5, perform tagmentation reaction at 37°C for 30 minutes.8. Add sodium dodecyl sulfate, proteinase K and heat at 55°C for 10 minutes to stop the tagmentation reaction and dissociate the TN5 from the DNA.9. Purify tagmented DNA using columns or beads.10. PCR amplify with the following PCR mix and protocol:11. 72°C for 5 minutes12. 98°C for 30 seconds13. 10-14 cycles of: a. 98°C for 10 seconds b. 63°C for 10 seconds14. 72°C for 1 minute15. Hold at 10°CNext generation sequencing, for example on an Illumina platform can be performed using paired-end sequencing with the following parameters:Read 1 : 38 base reads Read 2: 38 base reads Index 1 : 8 base reads Index 2: 16 base readsII. Recruitment of proteins, enzymes and complexes other than Tn5 and transposases and associated complexes[000119] The purpose of the recruited T n5 transposome is multifaceted in that it both fragments and inserts customizable transposons into the DNA proximal to the target location. This enables NGS readout to determine where the target of interest is located within the genome. Those skilled in the art would recognize that other transposases other than Tn5 could be used (sleeping beauty, TN7, TN3, TN 10, Vibrio harveyi, PiggBac, Hermes, Tol2). Additionally, those skilled in the art would recognize that recruitment of alternative proteins or molecules that are not used for tagmentation would also be useful. For example, enzymes that can modify the proximal DNA such as DNA nicking enzymes or restriction enzymes and other nucleases including MNase for CUT&RUN type applications of the method could be useful to fragment and / or tag the DNA or other nucleic acid in place of T n5, or non-fragmenting enzymes such as chromatin remodelers and epigenetic modifying enzymes (writers, readers or erasers) could be recruited to induce or detect a modification at the target site. Other useful enzymes include GC methylases and adenine methylases (e.g., EcoGII), which are useful in DNA methylation studies.[000120] The method / methods described herein for hybridization-based recruitment are amenable to recruitment of proteins / enzymes or molecules (referred to as the recruited element) other than Tn5 transposase. These alternate proteins / enzymes or molecules may require a different means of single stranded DNA or other hybridization means attachment using other chemical or molecular biology approaches including those know in the art and the examples below.1. Direct conjugation of the complement sequence of the antibody recruitment sequence to the recruitment element of interest through a sortase tag. If the recruitment element is a protein that does not bind transposons or other DNA sequences, a recombinant protein containing a sortase tag can be expressed. The same chemistry described in the “Antibody conjugation chemistry” section can be used to conjugate a sequence that is complementary to the antibody recruitment sequence that is conjugated on the recruitment element.Steps a. Express recombinant protein with sortase tag. b. Mix recombinant protein, sortase A5 enzyme and (Gly)5-Click label and incubate at 30°C for one hour with shaking. c. Remove excess label using column purification. d. Inactivate sortase. e. Mix the click moiety labeled recombinant protein with an oligonucleotide that has been labeled with the corresponding reactive click moiety. The labeled oligonucleotide contains the complement to the antibody recruitment sequence, and any other functional sequences such as indexes, NGS sequences or amplification sequences. f. Remove excess label using size exclusion or purification of choice. g. Incubate DNA conjugated primary antibody with sample. h. Wash in 150 mM NaCI to remove excess primary antibody. i. Incubate sample with DNA conjugated recombinant protein. j. Wash in appropriate buffer to remove excess recombinant protein. k. Bring reaction to conditions optimal for recombinant protein. This could be steps such as increasing salt concentration, adding a co-factor or changing the reaction temperature.2. Tn5 transposomes can be used as guide proteins for other proteins or molecules of interest, contingent on the recruited element not requiring magnesium for activation. Tn5 fusion proteins which contain a protein or molecule of interest to be recruited to the target site can be expressed and loaded with the customized transposons. The Tn5 transposome portion of the fusion protein will be recruited through hybridization between the antibody recruitment sequence and the transposon but will not be activated with magnesium. The other portion of the fusion protein can be activated by means such as adding a cofactor or changing the reaction temperature.[000121] Steps: a. Express a fusion protein of Tn5 and the recruited protein of interest. b. Load the fusion protein with transposons that contain the complementary DNA sequence to the antibody recruitment sequence. c. Incubate DNA conjugated primary antibody with sample. d. Wash in 150 mM NaCI to remove excess primary antibody.e. Incubate sample with loaded Tn5 fusion protein. f. Wash in 300 mM NaCI buffer to remove excess Tn5 fusion protein. g. Bring reaction to conditions optimal for the recruited protein / molecule. This could be steps such as increasing salt concentration, adding a co-factor or changing the reaction temperature.III. Multiplexed Recruitment[000122] The antibody recruitment sequence can be customized such that two or more recruitment sequences are conjugated directly to one antibody. This can be done using two methods:1. During the antibody conjugation step, two or more antibody recruitment sequences modified with the same click moiety are mixed with the antibody (FIG. 9A).2. The DNA sequence is designed such that one oligonucleotide contains two or more recruitment sequences. They may or may not be separated by a linker / spacer (FIG. 9B).[000123] In this embodiment the antibody or antibodies containing two or more antibody recruitment sequences could be incubated with a sample, followed by incubation with two or more Tn5 transposomes containing a complementary sequence to one of the antibody recruitment sequences and a unique index (FIG. 9). The transposomes could be added in parallel or sequentially. Here is described multiplexed recruitment of Tn5 transposomes, but alternative proteins that have been bound, loaded or modified such that they contain the complement DNA sequence to the antibody recruitment sequence could be used.[000124] Recruitment of more than one Tn5 transposome could be advantageous for collecting spatially resolved genomic data in a single cell. To achieve sequencing data with spatial resolution, two indexes, corresponding to two different physical locations such as axis, can be inserted into the genome at the target of interest and overlayed onto imaging data of the same sample. A slice of tissue could be first stained and imaged with multiple antibodies, each conjugated to multiple antibody recruitment sequences. Tn5 transposomes loaded with transposons containing the complementary sequence to one of the antibody recruitment sequences, and a unique index (Axis A) can be added to the sample on one axis (or other spatially resolved marker). This could be followed by the addition of a second Tn5 transposome with transposons containing a complementary sequence to the other recruitment sequence, and another unique index (Axis B) (or other spatially resolved marker).IV. Integration Into Single-Cell Multiomic Platform[000125] The collection of genomic, epigenomic and transcriptomic data within a single cell provides invaluable insight and requires a simple assay capable of multiplexing. The nature of the recruitment through hybridization strategy described enables ease of implementation intosingle cell platforms. Single cells can be isolated from native or fixed bulk samples such as tissue, bulk cells or organoids and can be individually profiled with antibodies for multiple targets. Prior to isolation, bulk samples can be incubated with multiple antibodies, each conjugated with a unique antibody recruitment sequence. Antibodies can be chosen such that multiomic data can be collected from each sample. After incubation with the antibodies, bulk cells can be isolated / separated / sorted into single cells using any platform amendable for single cell assays. Each cell with multiple antibodies bound at target locations will be isolated into a container / droplet, where Tn5 transposomes loaded with transposons containing a complementary sequence to a specific antibody recruitment sequence and functional sequences that are compatible with single cell profiling will be added. Such sequences in the transposon may include, in no specific order, 1) a cell specific index 2) UMI 3) target specific index 4) NGS flow cell binding and read sequences and 5) ME sequence. The resulting fragments after tagmentation and isolation can be bioinformatically deconvoluted to provide mapping information of each target, in each cell. The multiplexing capability of the hybridization recruitment strategy described enables collection of data at any location an antibody can bind within a sample, thus antibodies with epigenomic, genomic, or transcriptomic targets may be combined. Another method involves performing CUT&Tag reactions in bulk and then adding cell type-specific barcodes in droplets or picowells. Such methods can employ, for example, Both 10X™, Becton Dickinson Rhapsody™, and Fluent Biosciences™ platforms. Universal sequencing technology (UST) can be used in this context to improve the efficiency and accuracy of next-generation sequencing.V. Recruitment using pA-Tn5, pG-Tn5 or pAG-Tn5[000126] pA-Tn5, protein-G / Tn5 (pG-Tn5) or protein-A / protein-G / Tn5 (pAG-Tn5) can be loaded using the same method as Tn5, with transposons that contain customizable single stranded overhangs. In one embodiment, pA-Tn5, pG-Tn5 or pAG-Tn5 could be used to recruit Tn5 transposomes in place of recruitment directly by the antibody. In this design, pA-Tn5, pG- Tn5 or pAG-Tn5 could be loaded with transposons that contain a unique recruiting sequence in the single stranded overhang. In addition to the recruitment sequence, the transposon may also contain multiple functional sequences such as NGS flow cell binding and read sequences, unique indexes, and the ME sequence. Tn5 could then be loaded with transposons containing a sequence that is complementary to the recruiting sequence loaded into the pA-, pG- or pAG- Tn5 transposome. The pA-, pG- or pAG-Tn5 transposome could also contain other functional sequences such as NGS flow cell binding and read sequences, unique indexes and the ME sequence.[000127] There are two iterations of this embodiment, one for singleplex reactions and one for multiplexed reactions in which secondary antibodies are not required for either (FIG. 10). For singleplex reactions (FIG. 10A), a primary antibody will be incubated with a sample and bind toa target site. pA-, pG- or pAG-Tn5 transposomes loaded with transposons containing a recruitment sequence will be recruited to the target site through pA-, pG- or pAG-antibody binding. Tn5 transposomes loaded with transposons containing the complement to the recruitment sequence can then be recruited to the target site through hybridization, amplifying the amount of Tn5 transposomes without a secondary antibody. For multiplex reactions (FIG. 10B), multiple primary antibodies can be incubated with a pA-, pG- or pAG-Tn5 transposome that have been loaded with transposons containing a unique recruiting sequence, and a unique index, forming a pre-bound pA-, pG- or pAG-Tn5 / antibody complex that is unique for each target. The complex can then be incubated with the sample, binding at the target site. Tn5 transposomes loaded with transposons containing the complementary sequence to one of the recruitment sequences, and a unique index, can then be recruited specifically through hybridization.VI. Additional ExamplesA. Steps for singleplex reaction:[000128] Steps:1. Load pA-, pG-, or pAG-Tn5 with transposons that contain a unique recruiting sequence.2. Incubate the sample with a primary antibody.3. Wash in 150 mM NaCI wash buffer to remove excess primary antibody.4. Incubate sample with pA-, pG-, or pAG-Tn5 transposome.5. Wash in 300 mM NaCI wash buffer to remove excess pA-, pG-, or pAG-Tn5 transposome.6. Add TN5 containing complimentary hybridization sequence / s7. Add 10 mM MgCh to activate Tn5, perform tagmentation reaction at 37°C for 30 minutes.8. Add sodium dodecyl sulfate, proteinase K and heat at 55°C for 10 minutes to stop tagmentation reaction.9. Purify tagmented DNA using columns or beads.10. PCR amplify with the following PCR mix and protocol:11. 72°C for 5 minutes12. 98°C for 30 seconds13. 10-14 cycles of: a. 98°C for 10 seconds b. 63°C for 10 seconds14. 72°C for 1 minute15. Hold at 10°CNext generation sequencing on an Illumina platform can be performed using paired-end sequencing with the following parameters:Read 1 : 38 base readsRead 2: 38 base readsIndex 1: 8 base readsIndex 2: 16 base readsB. Steps for multiplexed reaction:[000129] Steps:1. Load pA-, pG-, or pAG-Tn5 with transposons that contain a unique recruiting sequence.2. Incubate each primary antibody with a uniquely loaded pA-, pG-, or pAG-Tn5 transposome.3. Incubate at room temperature for one hour.4. Incubate sample with the pre-bound pA-, pG-, or pAG-Tn5 / antibody complex.5. Wash in 300 mM NaCI wash buffer to remove excess pA-, pG-, or pAG-Tn5 / antibody complex.6. Add TN5 containing complimentary hybridization sequence / s7. Add 10 mM MgCh to activate Tn5, perform tagmentation reaction at 37°C for 30 minutes.8. Add sodium dodecyl sulfate, proteinase K and heat at 55°C for 10 minutes to stop the tagmentation reaction.9. Purify tagmented DNA using columns or beads.10. PCR amplify with the following PCR mix and protocol:11. 72°C for 5 minutes12. 98°C for 30 seconds13. 10-14 cycles of: a. 98°C for 10 seconds b. 63°C for 10 seconds14. 72°C for 1 minute15. Hold at 10°CNext generation sequencing on an Illumina platform can be performed using paired-end sequencing with the following parameters:Read 1 : 38 base readsRead 2: 38 base readsIndex 1: 8 base readsIndex 2: 16 base reads[000130] In another embodiment, a secondary antibody may be used to amplify the amount of pA-, pG- or pAG-Tn5 transposomes recruited to the target site (FIG. 11). Multiple primary antibodies, all of different animal species, can be incubated with the sample and bind at different target locations. Multiple secondary antibodies that recognize the different animal species canbe incubated with pA-, pG- or pAG-Tn5 transposomes loaded with the recruiting sequence and a unique index, forming pre-bound pA-, pG- or pAG-Tn5 / 2° antibody complexes, each with unique recruiting sequences and indexes. Each complex will contain a secondary antibody specific to a species of animal and a pA-, pG- or pAG-Tn5 transposome loaded with a transposon containing a unique recruitment sequence and index. The complexes can be incubated with the sample to bind each primary antibody. Tn5 transposomes each loaded with a unique transposon containing the complementary sequence to only one of the pA-, pG- or pAG- Tn5 recruitment sequences, and a unique index, can be incubated with the sample and recruited to the target site through hybridization. This example could also be accomplished using only one primary antibody and one animal species of secondary antibody, achieving singleplex detection.[000131] Steps:1. Load pA-, pG-, or pAG-Tn5 with transposons that contain a unique recruiting sequence.2. Incubate each secondary antibody with a uniquely loaded pA-, pG-, or pAG-Tn5 transposome.3. Incubate at room temperature for one hour.4. Incubate the sample with one or more primary antibodies. If using more than one primary antibody, they must be of different species.5. Wash in 150 mM NaCI wash buffer to remove excess primary antibody.6. Incubate sample with the pre-bound pA-, pG-, or pAG-Tn5 / secondary antibody complex.7. Wash in 300 mM NaCI wash buffer to remove excess pA-, pG-, or pAG-Tn5 / secondary antibody complex.8. Add TN5 containing complimentary hybridization sequence / s9. Add 10 mM MgCh to activate Tn5, perform tagmentation reaction at 37°C for 30 minutes.10. Add sodium dodecyl sulfate, proteinase K and heat at 55°C for 10 minutes to stop the tagmentation reaction.11. Purify tagmented DNA using columns or beads.12. PCR amplify with the following PCR mix and protocol:13. 72°C for 5 minutes14. 98°C for 30 seconds15. 14 cycles of: a. 98°C for 10 seconds b. 63°C for 10 seconds16. 72°C for 1 minute17. Hold at 10°CC. Pre-bound stable Antibody / Tn5 complexes[000132] The hybridization event that recruits Tn5 transposomes to the target antibody can also take place in solution prior to incubation with the sample. In this embodiment, the antibody / antibodies conjugated with a Tn5 recruitment oligonucleotide can be mixed with Tn5 transposomes that have been loaded with transposons containing the complementary sequence to the antibody recruitment sequence, in solution. This will form a stable association between each primary antibody and its specifically recruited Tn5 transposome through the complementary single stranded DNA regions. This complex can then be incubated with the sample where the antibody / Tn5 transposome complex will associate with the target through specific antibody recognition. The Tn5 transposome associated with the primary antibody is simultaneously recruited to the target site through this interaction and can be activated with an appropriate co-factor for site specific tagmentation. Since Tn5 transposomes will only associate with the intended antibody through specific hybridization, pre-bound antibody / Tn5 complexes with multiple primary antibodies can be formed either in a separate solution for each antibody, or in one solution. This embodiment is described using primary antibodies as the recruiting element and Tn5 transposomes as the recruited element, but those skilled in the art would recognize that this could be accomplished using any recruiting element and any recruited element.[000133] Steps:1. Conjugate a primary antibody with an oligonucleotide containing an antibody recruitment sequence.2. Load Tn5 with transposons containing the complimentary sequence to a specific antibody recruitment sequence.3. Incubate the DNA conjugated antibody with a Tn5 transposome loaded with transposons containing the complementary sequence to the antibody recruitment sequence.4. Incubate the sample with the antibody / Tn5 transposome complex or complexes.5. Wash in 300 mM NaCI wash buffer to remove excess antibody / Tn5 transposome complexes.6. Add 10 mM MgCh to active Tn5, perform tagmentation reaction at 37°C for 30 minutes.7. Add sodium dodecyl sulfate, proteinase K and heat at 55°C for 10 minutes to stop tagmentation reaction.8. Purify tagmented DNA using columns or beads.9. PCR amplify with the following PCR mix and protocol:10. 72°C for 5 minutes11. 98°C for 30 seconds12. 14 cycles of: a. 98°C for 10 seconds b. 63°C for 10 seconds13. 72°C for 1 minute14. Hold at 10°CVII. References (incorporated herein by reference):[000134] Buenrostro, J., Giresi, P, Zaba, L. et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10, 1213-1218 (2013). doi.org / 10.1038 / nmeth.2688[000135] Skene, P. J., Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. eLife 6:e21856 (2017). doi.org / 10.7554 / eLife.21856[000136] Kaya-Okur, H.S., Wu, S.J., Codomo, C.A. et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun 10, 1930 (2019). doi.org / 10.1038 / s41467-019-09982-5[000137] Gopalan, S., Wang, Y, Harper, N. W, Garber, M., Fazzio, T. G. Simultaneous profiling of multiple chromatin proteins in the same cells. Mol Cell 81, 4736-4746 (2021). doi.org / 10.1016 / j.molcel.2021.09.019[000138] Jelinek et al., Targeted Transposition For Use In Epigenetic Studies, patent application PCT / US2014 / 039250[000139] Fernandez et al., Multiplex Isolation of Protein-Associated Nucleic Acids, patent application PCT / US2012 / 066472EXEMPLARY EMOBDIMENTS[000140] 1. A method comprising: a) contacting chromatin with a recruitment complex comprising a binding agent bound to a recruitment nucleic acid molecule comprising a recruitment sequence, wherein the binding agent binds a target in the chromatin, and allowing the binding agent to bind the target; b) contacting the recruitment sequence with a modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence, and allowing the hybridization sequence to hybridize with the recruitment sequence; and c) allowing the nucleic acid modification enzyme to modify nucleic acid in the chromatin.[000141] 2. The method of embodiment 1, wherein contacting the chromatin with the binding agent is performed with intranuclear chromatin, intracellular chromatin, or extracellular chromatin.[000142] 3. The method of embodiment 1, wherein the chromatin is euchromatin or heterochromatin.[000143] 4. The method of embodiment 1, wherein the chromatin is fixed or non-fixed, or positioned on a solid support.[000144] 5. The method of embodiment 1, wherein the binding agent comprises an antibody; a protein that comprises a DNA binding domain (e.g., a bZIP domain, a helix-loop-helix, a helix- turn-helix, a leucine zipper, or a zinc finger); lexitropsin; a protein that interacts with chromatin (e.g., proteins comprising an MG-box, histone methylases, recruitment proteins, Swi proteins, or chromodomain or bromodomain); or a nucleic acid molecule (e.g., RNA or DNA) that has affinity for a specific target in DNA or RNA of chromatin.[000145] 6. The method of embodiment 1, wherein the binding agent specifically binds the target.[000146] 7. The method of embodiment 1, wherein the target is a histone comprising a post- translational modification (e.g., by methylation, acylation, phosphorylation, or ubiquination); a chromatin-associated protein (e.g., a chromatin remodeling protein or a polycomb group (PcG) protein); a topologically associating domain (TAD); a chromatin loop; a modified RNA; a modified DNA (e.g., methylated DNA), a cDNA, a transcription factor, a single stranded DNA binding protein; a DNA repair associated protein; a transcriptional machinery protein; a DNA replication associated protein; or a nucleotide sequence.[000147] 8. The method of embodiment 1, wherein the target is a DNA / RNA hybrid.[000148] 9. The method of embodiment 8, wherein the binding agent comprises antibody S9.6.[000149] 10. The method of embodiment 1, wherein the recruitment nucleic acid molecule comprises single stranded DNA, single stranded RNA, a modified nucleic acid, a phosphorothioate, an LNA, or a PNA.[000150] 11. The method of embodiment 1, wherein the recruitment nucleic acid molecule is covalently bound or non-covalently bound to the binding agent.[000151] 12. The method of embodiment 11, wherein the recruitment nucleic acid molecule is bound to the binding agent through an azide or alkyne linkage.[000152] 13. The method of embodiment 1, wherein the recruitment nucleic acid molecule comprises a plurality of the same recruitment sequences.[000153] 14. The method of embodiment 1, wherein the recruitment nucleic acid molecule comprises a plurality of different recruitment sequences.[000154] 15. The method of embodiment 14, comprising contacting the plurality of different recruitment sequences with a plurality of different complexes comprising different hybridization sequences, and allowing the different hybridization sequences to hybridize to the different recruitment sequences.[000155] 16. The method of embodiment 13 or 14, wherein the plurality of different recruitment sequences are separated by spacers.[000156] 17. The method of embodiment 1, wherein the recruitment nucleic acid molecule comprises a plurality of different recruitment sequences, and the method comprises contacting the binding agent with a plurality of different complexes comprising nucleic acid molecules comprising different hybridization sequences.[000157] 18. The method of embodiment 1, wherein the recruitment sequence comprises at least 10 nucleotides (e.g., at least 15 nucleotides or between 10 and 25 nucleotides, or between 18 and 25 nucleotides).[000158] 19. The method of embodiment 1, wherein the binding agent is bound to at least two recruitment nucleic acid molecules comprising the same recruitment sequences.[000159] 20. The method of embodiment 1, wherein the binding agent is bound to at least two different recruitment nucleic acid molecules comprising a different recruitment sequences.[000160] 21. The method of embodiment 1, wherein the nucleic acid modification enzyme comprises a transposase, a DNase, an MNase, a DNA nicking enzyme, a biotinylating enzyme, an endonuclease (e.g., a restriction enzyme, endonuclease I), an RNAse (e.g., RNase H), a topoisomerase, a reverse transcriptase, a polyA polymerase, a terminal transferase, Cas9, Cas12, TALENs, or T7.[000161] 22. The method of embodiment 1, wherein the hybridization sequence comprises at least 10 nucleotides (e.g., at least 15 nucleotides or between 10 and 25 nucleotides, or between 18 and 25 nucleotides).[000162] 23. The method of embodiment 1, wherein the hybridization sequence is fully complementary or partially complementary to the recruitment sequence.[000163] 24. The method of embodiment 1, wherein the complex comprises a transposome, wherein the transposome comprises first and second transposase molecules, wherein the first transposase molecule is loaded with a first transposon comprising a transposase recognition sequence and a 5’ overhang comprising the hybridization sequence, and the second transposase molecule is loaded with a second transposon comprising a transposase recognition sequence, optionally comprising a 5’ overhang comprising a second hybridization sequence (which may be the same sequence as the hybridization sequence of the first transposon).[000164] 25. The method of embodiment 24, wherein the first transposon further comprises one, two, three, four, or five of: a chemical moiety (e.g., biotin or a fluorescent label) a first flowcell binding sequence (e.g., functioning as an amplification priming sequence), a unique molecular identifier sequence, a first sample index sequence, and a primer binding sequence.[000165] 26. The method of embodiment 24, wherein the second transposon further comprises one two, three or four of: an amplification priming sequence a second flowcell binding sequence (e.g., functioning as an amplification priming sequence), unique molecular identifier sequence, a second sample index sequence, and a second primer binding sequence.[000166] 27. The method of embodiment 25 or 26, wherein the first and / or second flowcell binding sequences are selected from P5 and P7.[000167] 28. The method of embodiment 25 or 26, wherein the first and / or second sample index sequences are selected from i5 and i7.[000168] 29. The method of embodiment 24, wherein the transposons comprise sequences sufficient to support DNA amplification by any of PCR, rolling circle, Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Strand Displacement Amplification (SDA) Multiple Displacement Amplification (MDA), Nucleic Acid Sequence-Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA), and Hybridization Chain Reaction (HCR).[000169] 30. The method of embodiment 24, wherein the transposase comprises Tn5, sleeping beauty, TN7, TN3, TN10, Vibrio harveyi, PiggyBac, Hermes, or Tol2.[000170] 31. The method of embodiment 24, wherein the transposase recognition sequence comprises a terminal inverted repeat, a mosaic end sequence or a synthetic sequence.[000171] 32. The method of embodiment 24, wherein modifying the nucleic acid comprises cleaving the genomic DNA, attaching an adapter to a terminal end of cleaved DNA (optionally wherein the adapter comprises a chemical moiety, such as biotin or a fluorescent label), or cleaving RNA / DNA hybrids.[000172] 33. The method of embodiment 24, wherein modifying the nucleic acid produces a fragment of genomic DNA flanked by transposase recognition sequences.[000173] 34. The method of embodiment 33, wherein the fragment further comprises one or more of a pair of flowcell binding sequences (e.g., functioning as an amplification priming sequence), unique molecular identifier sequence, a pair of sample index sequences, and a pair of primer binding sequences.[000174] 35. The method of embodiment 1, wherein allowing the nucleic acid modification enzyme to modify nucleic acid in the chromatin comprising activating the nucleic acid modification enzyme.[000175] 36. The method of embodiment 35, wherein activating comprises exposing the nucleic acid modification enzyme to a metal ion (e.g., Mg++, Ca++, Mn++ Zn++, Co++, and Ni++).[000176] 37. The method of embodiment 35, wherein activating comprises exposing the nucleic acid modification enzyme to ATP, NADPH or a co-factor.[000177] 38. The method of any of embodiments 1-37, wherein:(I) the recruitment nucleic acid molecule comprises a plurality of the same or different hybridization sequences;(II) the method comprises contacting the plurality of recruitment sequences with one or more transposomes comprising first and second transposases bound to first and second transposons, wherein the first and second transposons comprise the same or different hybridization sequences, and wherein hybridization sequences of one transposon can hybridize with hybridization sequences of another transposon, whereby hybridization produces a chain or tree of modification complexes bound to hybridization.[000178] 39. The method of embodiment 38, wherein the recruitment complex comprises a plurality of recruitment nucleic acid molecules and the transposon comprises a plurality of the same or different hybridization sequences.[000179] 40. The method of any of embodiments 1-39, wherein the nucleic acid modification complex further comprises a recruitment nucleic acid molecule comprising a recruitment sequence, and the method further comprises contacting the modification complex with one or more secondary modification complexes, wherein each secondary modification complex comprises nucleic acid modification enzyme attached to a hybridization nucleic acid molecule comprising a hybridization sequence and a recruitment nucleic acid molecule comprising a recruitment sequence, wherein recruitment sequences can hybridize to hybridization sequences to form a chain of bound modification complexes.[000180] 41. The method of embodiment 40, wherein the modification complexes comprise transposomes loaded with a first transposon comprising a recruitment sequence and a second transposon comprising a hybridization sequence.[000181] 42. The method of any of embodiments 1-41, wherein:(I) the binding agent comprises a plurality of different binding agents bound to nucleic acid molecules having different recruitment sequences; and(II) the modification complex comprises a plurality of different modification complexes comprising nucleic acid molecules comprising different hybridization sequences, wherein the different hybridization sequences hybridize with the different recruitment sequences.[000182] 43. The method of any of embodiments 1-42, further comprising: d) amplifying the modified nucleic acid.[000183] 44. The method of embodiment 1, further comprising: sequencing the modified nucleic acid.[000184] 45. The method of any of embodiments 1-42, further comprising: d) isolating the modified nucleic acid; and, optionally, e) analyzing the isolated nucleic acid.[000185] 46. The method of embodiment 45, wherein analyzing comprises visually detecting binding sites in the chromatin, e.g., by fluorescent labeling.[000186] 47. The method of embodiment 45, wherein analyzing comprises sequencing the nucleic acid or mapping the nucleic acid to the genome.[000187] 48. The method of any of embodiments 44 to 47, wherein sequencing comprises high-throughput nucleic acid sequencing.[000188] 49. The method of any of embodiments 1 to 48, comprising: contacting the chromatin with the recruitment complex before contacting the recruitment sequence with a modification complex; or contacting the recruitment sequence with a modification complex before contacting the chromatin with the recruitment complex.[000189] 50. A multiplex method comprising: a) contacting chromatin with a plurality of different recruitment complexes comprising different binding agents bound to recruitment nucleic acid molecules comprising different recruitment sequences, wherein the binding agents bind different targets in the chromatin, and allowing the binding agents to bind the targets; b) contacting the different recruitment sequences with different modification complexes comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising different hybridization sequences, wherein different hybridization sequences hybridize to different recruitment sequences, and allowing the hybridization sequences to hybridize with the recruitment sequences; and c) allowing the nucleic acid modification enzymes to modify nucleic acid in the chromatin.[000190] 51. The method of embodiment 50, wherein the different modification complexes comprise transposomes comprising first and second transposases, wherein at least the first transposases are loaded with transposons comprising transposase recognition sequences and a 5’ overhang comprising the different hybridization sequences.[000191] 52. The method of embodiment 51, wherein each different hybridization sequence is paired with a different molecular identifying nucleotide sequence on the transposon bound to the first or second transposase.[000192] 53. The method of embodiment 52, wherein the different binding agents bind different histone modifications, different transcription factors or different histone modifying enzymes.[000193] 54. A kit comprising: a) one or more containers, wherein a plurality of the containers each contain a recruitment complex (or containers containing components of a recruitment complex), wherein each recruitment complex comprises a different binding agent bound to a recruitment nucleic acid molecule comprising a different hybridization sequence; and b) one or more containers, wherein a plurality of the containers each contain a modification complex (or containers containing components of a modification complex) comprising a same or different nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a different hybridization sequence that hybridizes with a different recruitment sequence.[000194] 55. The kit of embodiment 54, further comprising one or more containers selected from:(I) a container containing a nucleic acid modification enzyme activating agent;(II) a container containing a buffer comprising a detergent;(III) a container containing concavalin A beads;(IV) a container containing a binding buffer;(V) a container containing a wash buffer;(VI) a container containing Antibody buffer(VII) a container containing Protease inhibitor(VIII) a container containing Stop buffer;(IX) a container containing Proteinase K;(X) a container containing a solid phase for DNA capture;(XI) a container containing dNTPs, amplification primers, and polymerase;(XII) a container containing SPRI or other beads for purification of DNA after PCR; and(XIII) a container containing a reagent for coupling a nucleic acid molecule to a protein.[000195] 56. The kit of embodiment 54 or 55, wherein at least one of the binding agents comprises an antibody.[000196] 57. The kit of embodiment 54 to 56, wherein at least one of the modification complexes comprises a transposome comprising first and second transposases, each transposase bound to a transposon comprising a transposase recognition sequence, wherein at least one of the transposons comprises the hybridization sequence.[000197] 58. A modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence.[000198] 59. The modification complex of embodiment 58, wherein the complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein the first transposase is loaded with a first transposon comprising a transposase recognition sequence and a 5’ overhang comprising a hybridization sequence.[000199] 60. The modification complex of embodiment 59, wherein the first transposon comprises one two, three or four or five of: a chemical moiety (e.g., biotin or a fluorescent label, a first flowcell binding sequence (e.g., functioning as an amplification priming sequence), unique molecular identifier sequence, a first sample index sequence, and a first primer binding sequence.[000200] 61. The modification complex of embodiment 60, wherein the second transposase comprises one, two, three or four of: an amplification priming sequence a second flowcell binding sequence (e.g., functioning as an amplification priming sequence), unique molecular identifier sequence, a second sample index sequence, and a second primer binding sequence.[000201] 62. A complex comprising: a) chromatin comprising a target; b) a recruitment complex comprising a binding agent bound to a recruitment nucleic acid molecule comprising a recruitment sequence, wherein the binding agent binds a target in the chromatin, wherein the binding agent is bound to the target; c) a modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence, wherein the hybridization sequence is hybridized with the recruitment sequence.[000202] 63. The complex of embodiment 62, wherein the binding agent comprises an antibody.[000203] 64. The complex of embodiment 62 or 63, wherein the complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein the first transposase is loaded with a first transposon comprising a transposase recognition sequence and a 5’ overhang comprising a hybridization sequence.[000204] 65. A method comprising: a) providing a complex of any of embodiments 62 to 64; andb) activating the nucleic acid modification enzyme.[000205] 66. A method of making transposome comprising: a) providing a first transposon comprising a double stranded transposase recognition sequence and a single stranded 5’ overhang comprising a hybridization sequence; and a second transposon comprising a double stranded transposase recognition sequence; and b) incubating the first and second transposons with transposase molecules; whereby the transposons associate with transposase molecules, and the transposase molecules dimerize to form the transposome.[000206] 67. The method of embodiment 66, wherein a transposase is comprised in a fusion molecule comprising protein A or protein G or protein A / G or other Fc region binding protein.[000207] 68. A method comprising: a) contacting chromatin with a binding agent that binds a target in the chromatin, and allowing the binding agent to bind the target; b) contacting the binding agent with a primary modification complex, wherein the primary modification complex comprises:(I) a nucleic acid modification enzyme bound to a recruitment nucleic acid molecule comprising a recruitment sequence;(II) an affinity agent having binding affinity for the binding agent, and allowing the affinity agent to bind the binding agent; c) contacting the recruitment sequence with a secondary modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence, and allowing the hybridization sequence to hybridize with the recruitment sequence; and d) allowing the nucleic acid modification enzyme or enzymes to modify nucleic acid in the chromatin.69. The method of embodiment 68, wherein:(I) the primary modification complex comprises the affinity agent bound to a transposome, wherein the transposome comprises first and second transposases, wherein at least the first transposase is loaded with a transposon comprising a transposase recognition sequence a 5’ overhang comprising the recruitment sequence; and(II) the secondary modification complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein at least the first transposase is loaded with a transposon comprising a transposase recognition sequence and a 5’ overhang comprising the hybridization sequence.[000208] 70. The method of embodiment 68 or 69, comprising: contacting the primary modification complex with the binding agent before contacting the binding agent with the chromatin; orcontacting the binding agent with the chromatin before contacting the primary modification complex with the binding agent.[000209] 71 . The method of embodiment 70, wherein the binding agent comprises an antibody.[000210] 72. The method of embodiment 71 , wherein the affinity agent comprises protein A or protein G.[000211] 73. The method of embodiment 72, wherein the affinity agent comprises a fusion protein comprising the transposase and protein A or protein G.[000212] 74. The method of embodiment 70, wherein the affinity agent comprises a terminal affinity element having affinity for the binding agent, and an intermediate affinity element having affinity for the terminal affinity element.[000213] 75. The method of embodiment 74, wherein the binding agent comprises a primary antibody, the terminal affinity agent comprises an antibody with affinity for the primary antibody, and the intermediate affinity element comprises protein A or protein G bound to the primary affinity element.[000214] 76. The method of embodiment 75, wherein the affinity agent comprises a fusion protein comprising a transposase and protein A or protein G.[000215] 77. The method of any of embodiments 68-76, wherein the nucleic acid modification complex further comprises a recruitment nucleic acid molecule comprising a recruitment sequence, and the method further comprises contacting the modification complex with one or more secondary modification complexes, wherein each secondary modification complex comprises nucleic acid modification enzyme attached to a hybridization nucleic acid molecule comprising a hybridization sequence and a recruitment nucleic acid molecule comprising a recruitment sequence, wherein recruitment sequences can hybridize to hybridization sequences to form a chain of bound modification complexes.[000216] 78. The method of embodiment 77, wherein the modification complexes comprise transposomes loaded with a first transposon comprising a recruitment sequence and a second transposon comprising a hybridization sequence.[000217] 79. The method of any of embodiments 68-76, further comprising:[000218] amplifying; and, optionally, sequencing the modified nucleic acid.[000219] 80. A multiplex method comprising: a) providing a plurality of binding agent-modification complexes, wherein a plurality of the binding agent-modification complexes each comprise a different binding agent having affinity for a different target, bound to at least one primary modification complex comprising:(I) an affinity agent having binding affinity for the binding agent, and(II) a nucleic acid modification enzyme bound to a recruitment nucleic acid molecule comprising a different recruitment sequence, wherein the modification complex is bound to the binding agent through the affinity agent; b) contacting the plurality of binding agent-modification complexes with chromatin, and allowing the binding agents to bind their respective targets in the chromatin; c) contacting the different recruitment sequences with a plurality of secondary modification complexes, wherein a plurality of the secondary modification complexes each comprise a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a different hybridization sequence, wherein different hybridization sequences are adapted to hybridize to different recruitment sequences, and allowing the different a hybridization sequences to hybridize with the different recruitment sequences; and d) allowing the nucleic acid modification enzyme or enzymes to modify nucleic acid in the chromatin.[000220] 81. The method of embodiment 80, wherein:(I) the primary modification complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein the first and second transposases are loaded with a transposon comprising a transposase recognition sequence and a 5’ overhang comprising the recruitment sequence; and(II) the secondary modification complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein at least the first transposase is loaded with a transposon comprising a transposase recognition sequence and a 5’ overhang comprising the hybridization sequence.[000221] 82. The method of any of embodiments 80-81 , further comprising: e) amplifying and, optionally, sequencing, the isolated nucleic acid.[000222] 83. A multiplex method comprising: a) contacting chromatin with a plurality of different binding agents, wherein each of a plurality of the different binding agents binds to different targets in the chromatin, and allowing the binding agent to bind the target; b) contacting the binding agents with a plurality of different primary modification complexes, wherein each of a plurality of the different primary modification complex comprises:(I) a nucleic acid modification enzyme bound to a recruitment nucleic acid molecule comprising a different recruitment sequence;(II) an affinity agent having binding affinity for a different binding agent, and allowing the affinity agents to bind the binding agents; c) contacting the recruitment sequences with a plurality of different secondary modification complexes, wherein each of a plurality of the different secondary modificationcomplexes comprises a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a different hybridization sequence, wherein the different hybridization sequences hybridize to different recruitment sequences; and allowing the hybridization sequences to hybridize with the recruitment sequences; and d) allowing the nucleic acid modification enzyme or enzymes to modify nucleic acid in the chromatin.[000223] 84. The method of embodiment 83, wherein the affinity agents comprise different secondary affinity agents having affinity for the different binding agents, and an intermediate affinity element having affinity for the secondary affinity agent.[000224] 85. The method of embodiment 84, wherein the binding agents comprise a primary antibody, the secondary affinity agents comprise antibodies with affinity for the different primary antibodies, and the intermediate affinity elements comprise protein A or protein G bound to the primary affinity element.[000225] 86. The method of embodiment 85, wherein the affinity agent comprises a fusion protein comprising a transposase and protein A or protein G.[000226] 87. The method of embodiment 80, wherein the different first modification complexes comprise transposomes comprising first and second transposases, wherein the first transposases are loaded with transposons comprising transposase recognition sequences and a 5’ overhang comprising the different recruitment sequences.[000227] 88. The method of embodiment 87, wherein the different second modification complexes comprise transposomes comprising first and second transposases, wherein the first transposases are loaded with transposons comprising transposase recognition sequences and a 5’ overhang comprising the different recruitment sequences.[000228] 89. The method of any of embodiments 83-88, wherein the primary antibodies comprise antibodies of different animal species.[000229] 90. The method of any of embodiments 83-89, wherein the secondary binding agents comprise secondary antibodies.[000230] 91. The method of embodiment 90, wherein different secondary antibodies recognize antibodies of different species.[000231] 92. The method of embodiment 90, wherein the first complexes are bound to the secondary binding agents through protein A or protein G.[000232] 93. The method of embodiment 92, wherein protein A or protein G are comprised in a fusion protein with a transposase.[000233] 94. The method of any of embodiments 83-93, further comprising: e) amplifying and, optionally, sequencing, the isolated nucleic acid.[000234] 95. The method of any of embodiments 80-93, further comprising: d) isolating the modified nucleic acid; and, optionally, e) analyzing the isolated nucleic acid.[000235] 96. A method for detecting or mapping nucleic acid and nucleic acid-associated features of interest comprising: contacting said feature with a binding agent that specifically recognizes the feature of interest, wherein the binding agent is coupled to a first hybridization means; adding at least one nucleic acid modification means coupled to (i) at least one second hybridization means complementary to the first hybridization means and (ii) a nucleic acid sequencing or isolation means; allowing the first hybridization means to hybridize to the at least one second complementary hybridization means; activating the nucleic acid modification means to modify the nucleic acid proximal to the feature of interest; isolating or sequencing the modified nucleic acid using the sequencing or isolation means, thereby mapping or detecting the location in the nucleic acid of the feature of interest.[000236] 97. The method of embodiment 96, wherein: a) the binding agent comprises an antibody or other binding protein b) the hybridization means comprises single-strand oligonucleotides c) the nucleic acid modification means comprises a transposase d) the nucleic acid sequencing or isolation means comprises a priming adaptor[000237] 98. The method of embodiments 96 or 97, wherein the nucleic acid modification means comprises a DNase, an MNase (which digests single-stranded DNA (ssDNA), doublestranded DNA (dsDNA), and RNA with a preference for AT-rich sequences), a DNA nicking enzyme, a biotinylating enzyme, an endonuclease (e.g., a restriction enzyme, endonuclease I), an RNAse (e.g., RNase H), a topoisomerase, a reverse transcriptase, a polyA polymerase, a terminal transferase, Cas9, Cas12, TALENs, or T7.[000238] 99. The method of embodiments 96-98 wherein the nucleic acid modification means coupled to the second hybridization means further comprises at least one additional hybridization means for hybridizing to and recruiting further nucleic acid modification means coupled to hybridization means complementary to said at least one additional hybridization means.[000239] 100. A complex for detecting or mapping nucleic acid and nucleic acid-associated features of interest comprising: a) a binding agent that specifically recognizes a nucleic acid or nucleic acid associated feature of interest, wherein the binding agent is coupled to a first hybridization means; b) at least one nucleic acid modification means coupled to (i) at least one second hybridization means complementary to the first hybridization means and (ii) a nucleic acid sequencing or isolation means[000240] 101. A complex for detecting or mapping nucleic acid and nucleic acid-associated features of interest comprising the complex of embodiment 100, plus at least one additional hybridization means for hybridizing to and recruiting further nucleic acid modification means coupled to said at least one additional hybridization means.[000241] As used herein, the following meanings apply unless otherwise specified. The words “can” and “may” are used in a permissive sense (i.e. , meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words “include”, “including”, and “includes” and the like mean including, but not limited to. The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.” The phrase “at least one” includes “one”, “one or more”, “one or a plurality”, and, therefore, contemplates the use of the term “a plurality”. The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1 , 2 or 3” means “at least 1 , at least 2 or at least 3”. The term “about” refers to a range that is 5% plus or minus from a stated numerical value within the context of the particular usage. The term "consisting essentially of" refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination.[000242] It should be understood that the description and the drawings are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed oromitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims.[000243] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method comprising: a) contacting chromatin with a recruitment complex comprising a binding agent bound to a recruitment nucleic acid molecule comprising a recruitment sequence, wherein the binding agent binds a target in the chromatin, and allowing the binding agent to bind the target; b) contacting the recruitment sequence with a modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence, and allowing the hybridization sequence to hybridize with the recruitment sequence; and c) allowing the nucleic acid modification enzyme to modify nucleic acid in the chromatin.

2. The method of claim 1 , wherein contacting the chromatin with the binding agent is performed with intranuclear chromatin, intracellular chromatin, or extracellular chromatin.

3. The method of claim 1 , wherein the chromatin is euchromatin or heterochromatin.

4. The method of claim 1 , wherein the chromatin is fixed or non-fixed, or positioned on a solid support.

5. The method of claim 1 , wherein the binding agent comprises an antibody; a protein that comprises a DNA binding domain (e.g., a bZIP domain, a helix-loop-helix, a helix- turn-helix, a leucine zipper, or a zinc finger); lexitropsin; a protein that interacts with chromatin (e.g., proteins comprising an MG-box, histone methylases, recruitment proteins, Swi proteins, or chromodomain or bromodomain); or a nucleic acid molecule (e.g., RNA or DNA) that has affinity for a specific target in DNA or RNA of chromatin.

6. The method of claim 1, wherein the binding agent specifically binds the target.

7. The method of claim 1, wherein the target is a histone comprising a post- translational modification (e.g., by methylation, acylation, phosphorylation, or ubiquination); a chromatin-associated protein (e.g., a chromatin remodeling protein or a polycomb group (PcG) protein); a topologically associating domain (TAD); a chromatin loop; a modified RNA; a modified DNA (e.g., methylated DNA), a cDNA, a transcription factor, a single stranded DNA binding protein; a DNA repair associated protein; a transcriptional machinery protein; a DNA replication associated protein; or a nucleotide sequence.

8. The method of claim 1 , wherein the target is a DNA / RNA hybrid.

9. The method of claim 8, wherein the binding agent comprises antibody S9.6.

10. The method of claim 1, wherein the recruitment nucleic acid molecule comprises single stranded DNA, single stranded RNA, a modified nucleic acid, a phosphorothioate, an LNA, or a PNA.

11. The method of claim 1 , wherein the recruitment nucleic acid molecule is covalently bound or non-covalently bound to the binding agent.

12. The method of claim 11, wherein the recruitment nucleic acid molecule is bound to the binding agent through an azide or alkyne linkage.

13. The method of claim 1, wherein the recruitment nucleic acid molecule comprises a plurality of the same recruitment sequences.

14. The method of claim 1, wherein the recruitment nucleic acid molecule comprises a plurality of different recruitment sequences.

15. The method of claim 14, comprising contacting the plurality of different recruitment sequences with a plurality of different complexes comprising different hybridization sequences, and allowing the different hybridization sequences to hybridize to the different recruitment sequences.

16. The method of claim 13 or 14, wherein the plurality of different recruitment sequences are separated by spacers.

17. The method of claim 1, wherein the recruitment nucleic acid molecule comprises a plurality of different recruitment sequences, and the method comprises contacting the binding agent with a plurality of different complexes comprising nucleic acid molecules comprising different hybridization sequences.

18. The method of claim 1 , wherein the recruitment sequence comprises at least 10 nucleotides (e.g., at least 15 nucleotides or between 10 and 25 nucleotides, or between 18 and 25 nucleotides).

19. The method of claim 1 , wherein the binding agent is bound to at least two recruitment nucleic acid molecules comprising the same recruitment sequences.

20. The method of claim 1 , wherein the binding agent is bound to at least two different recruitment nucleic acid molecules comprising a different recruitment sequences.

21. The method of claim 1 , wherein the nucleic acid modification enzyme comprises a transposase, a DNase, an MNase, a DNA nicking enzyme, a biotinylating enzyme, an endonuclease (e.g., a restriction enzyme, endonuclease I), an RNAse (e.g., RNase H), a topoisomerase, a reverse transcriptase, a polyA polymerase, a terminal transferase, Cas9, Cas12, TALENs, or T7.

22. The method of claim 1 , wherein the hybridization sequence comprises at least 10 nucleotides (e.g., at least 15 nucleotides or between 10 and 25 nucleotides, or between 18 and 25 nucleotides).

23. The method of claim 1 , wherein the hybridization sequence is fully complementary or partially complementary to the recruitment sequence.

24. The method of claim 1 , wherein the complex comprises a transposome, wherein the transposome comprises first and second transposase molecules, wherein the first transposase molecule is loaded with a first transposon comprising a transposase recognition sequence and a 5’ overhang comprising the hybridization sequence, and the second transposase molecule is loaded with a second transposon comprising a transposase recognitionsequence, optionally comprising a 5’ overhang comprising a second hybridization sequence (which may be the same sequence as the hybridization sequence of the first transposon).

25. The method of claim 24, wherein the first transposon further comprises one, two, three, four, or five of: a chemical moiety (e.g., biotin or a fluorescent label) a first flowcell binding sequence (e.g., functioning as an amplification priming sequence), a unique molecular identifier sequence, a first sample index sequence, and a primer binding sequence.

26. The method of claim 24, wherein the second transposon further comprises one two, three or four of: an amplification priming sequence a second flowcell binding sequence (e.g., functioning as an amplification priming sequence), unique molecular identifier sequence, a second sample index sequence, and a second primer binding sequence.

27. The method of claim 25 or 26, wherein the first and / or second flowcell binding sequences are selected from P5 and P7.

28. The method of claim 25 or 26, wherein the first and / or second sample index sequences are selected from i5 and i7.

29. The method of claim 24, wherein the transposons comprise sequences sufficient to support DNA amplification by any of PCR, rolling circle, Loop-Mediated Isothermal Amplification (LAMP), Helicase-Dependent Amplification (HDA), Strand Displacement Amplification (SDA) Multiple Displacement Amplification (MDA), NucleicAcid Sequence-Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA), and Hybridization Chain Reaction (HCR).

30. The method of claim 24, wherein the transposase comprises Tn5, sleeping beauty, TN7, TN3, TN10, Vibrio harveyi, PiggyBac, Hermes, or Tol2.

31. The method of claim 24, wherein the transposase recognition sequence comprises a terminal inverted repeat, a mosaic end sequence or a synthetic sequence.

32. The method of claim 24, wherein modifying the nucleic acid comprises cleaving the genomic DNA, attaching an adapter to a terminal end of cleaved DNA (optionally wherein the adapter comprises a chemical moiety, such as biotin or a fluorescent label), or cleaving RNA / DNA hybrids.

33. The method of claim 24, wherein modifying the nucleic acid produces a fragment of genomic DNA flanked by transposase recognition sequences.

34. The method of claim 33, wherein the fragment further comprises one or more of a pair of flowcell binding sequences (e.g., functioning as an amplification priming sequence), unique molecular identifier sequence, a pair of sample index sequences, and a pair of primer binding sequences.

35. The method of claim 1 , wherein allowing the nucleic acid modification enzyme to modify nucleic acid in the chromatin comprising activating the nucleic acid modification enzyme.

36. The method of claim 35, wherein activating comprises exposing the nucleic acid modification enzyme to a metal ion (e.g., Mg++, Ca++, Mn++Zn++, Co++, and Ni++).

37. The method of claim 35, wherein activating comprises exposing the nucleic acid modification enzyme to ATP, NADPH or a co-factor.

38. The method of any of claims 1-37, wherein:(I) the recruitment nucleic acid molecule comprises a plurality of the same or different hybridization sequences;(II) the method comprises contacting the plurality of recruitment sequences with one or more transposomes comprising first and second transposases bound to first and second transposons, wherein the first and second transposons comprise the same or different hybridization sequences, and wherein hybridization sequences of one transposon can hybridize with hybridization sequences of another transposon, whereby hybridization produces a chain or tree of modification complexes bound to hybridization.

39. The method of claim 38, wherein the recruitment complex comprises a plurality of recruitment nucleic acid molecules and the transposon comprises a plurality of the same or different hybridization sequences.

40. The method of any of claims 1-39, wherein the nucleic acid modification complex further comprises a recruitment nucleic acid molecule comprising a recruitment sequence, and the method further comprises contacting the modification complex with one or more secondary modification complexes, wherein each secondary modification complex comprises nucleic acid modification enzyme attached to a hybridization nucleic acid molecule comprising a hybridization sequence and a recruitment nucleic acid molecule comprising a recruitment sequence, wherein recruitment sequences can hybridize to hybridization sequences to form a chain of bound modification complexes.

41. The method of claim 40, wherein the modification complexes comprise transposomes loaded with a first transposon comprising a recruitment sequence and a second transposon comprising a hybridization sequence.

42. The method of any of claims 1-41, wherein:(I) the binding agent comprises a plurality of different binding agents bound to nucleic acid molecules having different recruitment sequences; and(II) the modification complex comprises a plurality of different modification complexes comprising nucleic acid molecules comprising different hybridization sequences, wherein the different hybridization sequences hybridize with the different recruitment sequences.

43. The method of any of claims 1-42, further comprising: d) amplifying the modified nucleic acid.

44. The method of claim 1 , further comprising: sequencing the modified nucleic acid.

45. The method of any of claims 1-42, further comprising: d) isolating the modified nucleic acid; and, optionally, e) analyzing the isolated nucleic acid.

46. The method of claim 45, wherein analyzing comprises visually detecting binding sites in the chromatin, e.g., by fluorescent labeling.

47. The method of claim 45, wherein analyzing comprises sequencing the nucleic acid or mapping the nucleic acid to the genome.

48. The method of any of claims 44 to 47, wherein sequencing comprises high- throughput nucleic acid sequencing.

49. The method of any of claims 1 to 48, comprising: contacting the chromatin with the recruitment complex before contacting the recruitment sequence with a modification complex; or contacting the recruitment sequence with a modification complex before contacting the chromatin with the recruitment complex.

50. A multiplex method comprising: a) contacting chromatin with a plurality of different recruitment complexes comprising different binding agents bound to recruitment nucleic acid molecules comprising different recruitment sequences, wherein the binding agents bind different targets in the chromatin, and allowing the binding agents to bind the targets; b) contacting the different recruitment sequences with different modification complexes comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising different hybridization sequences, wherein different hybridization sequences hybridize to different recruitment sequences, and allowing the hybridization sequences to hybridize with the recruitment sequences; and c) allowing the nucleic acid modification enzymes to modify nucleic acid in the chromatin.

51. The method of claim 50, wherein the different modification complexes comprise transposomes comprising first and second transposases, wherein at least the first transposases are loaded with transposons comprising transposase recognition sequences and a 5’ overhang comprising the different hybridization sequences.

52. The method of claim 51 , wherein each different hybridization sequence is paired with a different molecular identifying nucleotide sequence on the transposon bound to the first or second transposase.

53. The method of claim 52, wherein the different binding agents bind different histone modifications, different transcription factors or different histone modifying enzymes.

54. A kit comprising: a) one or more containers, wherein a plurality of the containers each contain a recruitment complex (or containers containing components of a recruitment complex), whereineach recruitment complex comprises a different binding agent bound to a recruitment nucleic acid molecule comprising a different hybridization sequence; and b) one or more containers, wherein a plurality of the containers each contain a modification complex (or containers containing components of a modification complex) comprising a same or different nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a different hybridization sequence that hybridizes with a different recruitment sequence.

55. The kit of claim 54, further comprising one or more containers selected from:(I) a container containing a nucleic acid modification enzyme activating agent;(II) a container containing a buffer comprising a detergent;(III) a container containing concavalin A beads;(IV) a container containing a binding buffer;(V) a container containing a wash buffer;(VI) a container containing Antibody buffer(VII) a container containing Protease inhibitor(VIII) a container containing Stop buffer;(IX) a container containing Proteinase K;(X) a container containing a solid phase for DNA capture;(XI) a container containing dNTPs, amplification primers, and polymerase;(XII) a container containing SPRI or other beads for purification of DNA after PCR; and(XIII) a container containing a reagent for coupling a nucleic acid molecule to a protein.

56. The kit of claim 54 or 55, wherein at least one of the binding agents comprises an antibody.

57. The kit of claim 54 to 56, wherein at least one of the modification complexes comprises a transposome comprising first and second transposases, each transposase bound to a transposon comprising a transposase recognition sequence, wherein at least one of the transposons comprises the hybridization sequence.

58. A modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence.

59. The modification complex of claim 58, wherein the complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein the first transposase is loaded with a first transposon comprising a transposase recognition sequence and a 5’ overhang comprising a hybridization sequence.

60. The modification complex of claim 59, wherein the first transposon comprises one two, three or four or five of: a chemical moiety (e.g., biotin or a fluorescent label, a first flowcell binding sequence (e.g., functioning as an amplification priming sequence), uniquemolecular identifier sequence, a first sample index sequence, and a first primer binding sequence.

61. The modification complex of claim 60, wherein the second transposase comprises one, two, three or four of: an amplification priming sequence a second flowcell binding sequence (e.g., functioning as an amplification priming sequence), unique molecular identifier sequence, a second sample index sequence, and a second primer binding sequence.

62. A complex comprising: a) chromatin comprising a target; b) a recruitment complex comprising a binding agent bound to a recruitment nucleic acid molecule comprising a recruitment sequence, wherein the binding agent binds a target in the chromatin, wherein the binding agent is bound to the target; c) a modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence, wherein the hybridization sequence is hybridized with the recruitment sequence.

63. The complex of claim 62, wherein the binding agent comprises an antibody.

64. The complex of claim 62 or 63, wherein the complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein the first transposase is loaded with a first transposon comprising a transposase recognition sequence and a 5’ overhang comprising a hybridization sequence.

65. A method comprising: a) providing a complex of any of claims 62 to 64; and b) activating the nucleic acid modification enzyme.

66. A method of making transposome comprising: a) providing a first transposon comprising a double stranded transposase recognition sequence and a single stranded 5’ overhang comprising a hybridization sequence; and a second transposon comprising a double stranded transposase recognition sequence; and b) incubating the first and second transposons with transposase molecules; whereby the transposons associate with transposase molecules, and the transposase molecules dimerize to form the transposome.

67. The method of claim 66, wherein a transposase is comprised in a fusion molecule comprising protein A or protein G or protein A / G or other Fc region binding protein.

68. A method comprising: a) contacting chromatin with a binding agent that binds a target in the chromatin, and allowing the binding agent to bind the target; b) contacting the binding agent with a primary modification complex, wherein the primary modification complex comprises:(I) a nucleic acid modification enzyme bound to a recruitment nucleic acid molecule comprising a recruitment sequence;(II) an affinity agent having binding affinity for the binding agent, and allowing the affinity agent to bind the binding agent; c) contacting the recruitment sequence with a secondary modification complex comprising a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a hybridization sequence, and allowing the hybridization sequence to hybridize with the recruitment sequence; and d) allowing the nucleic acid modification enzyme or enzymes to modify nucleic acid in the chromatin.

69. The method of claim 68, wherein:(I) the primary modification complex comprises the affinity agent bound to a transposome, wherein the transposome comprises first and second transposases, wherein at least the first transposase is loaded with a transposon comprising a transposase recognition sequence a 5’ overhang comprising the recruitment sequence; and(II) the secondary modification complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein at least the first transposase is loaded with a transposon comprising a transposase recognition sequence and a 5’ overhang comprising the hybridization sequence.

70. The method of claim 68 or 69, comprising: contacting the primary modification complex with the binding agent before contacting the binding agent with the chromatin; or contacting the binding agent with the chromatin before contacting the primary modification complex with the binding agent.

71. The method of claim 70, wherein the binding agent comprises an antibody.

72. The method of claim 71 , wherein the affinity agent comprises protein A or protein G.

73. The method of claim 72, wherein the affinity agent comprises a fusion protein comprising the transposase and protein A or protein G.

74. The method of claim 70, wherein the affinity agent comprises a terminal affinity element having affinity for the binding agent, and an intermediate affinity element having affinity for the terminal affinity element.

75. The method of claim 74, wherein the binding agent comprises a primary antibody, the terminal affinity agent comprises an antibody with affinity for the primary antibody, and the intermediate affinity element comprises protein A or protein G bound to the primary affinity element.

76. The method of claim 75, wherein the affinity agent comprises a fusion protein comprising a transposase and protein A or protein G.

77. The method of any of claims 68-76, wherein the nucleic acid modification complex further comprises a recruitment nucleic acid molecule comprising a recruitmentsequence, and the method further comprises contacting the modification complex with one or more secondary modification complexes, wherein each secondary modification complex comprises nucleic acid modification enzyme attached to a hybridization nucleic acid molecule comprising a hybridization sequence and a recruitment nucleic acid molecule comprising a recruitment sequence, wherein recruitment sequences can hybridize to hybridization sequences to form a chain of bound modification complexes.

78. The method of claim 77, wherein the modification complexes comprise transposomes loaded with a first transposon comprising a recruitment sequence and a second transposon comprising a hybridization sequence.

79. The method of any of claims 68-76, further comprising: amplifying; and, optionally, sequencing the modified nucleic acid.

80. A multiplex method comprising: a) providing a plurality of binding agent-modification complexes, wherein a plurality of the binding agent-modification complexes each comprise a different binding agent having affinity for a different target, bound to at least one primary modification complex comprising:(I) an affinity agent having binding affinity for the binding agent, and(II) a nucleic acid modification enzyme bound to a recruitment nucleic acid molecule comprising a different recruitment sequence, wherein the modification complex is bound to the binding agent through the affinity agent; b) contacting the plurality of binding agent-modification complexes with chromatin, and allowing the binding agents to bind their respective targets in the chromatin; c) contacting the different recruitment sequences with a plurality of secondary modification complexes, wherein a plurality of the secondary modification complexes each comprise a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a different hybridization sequence, wherein different hybridization sequences are adapted to hybridize to different recruitment sequences, and allowing the different a hybridization sequences to hybridize with the different recruitment sequences; and d) allowing the nucleic acid modification enzyme or enzymes to modify nucleic acid in the chromatin.

81. The method of claim 80, wherein:(I) the primary modification complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein the first and second transposases are loaded with a transposon comprising a transposase recognition sequence and a 5’ overhang comprising the recruitment sequence; and(II) the secondary modification complex comprises a transposome, wherein the transposome comprises first and second transposases, wherein at least the first transposase isloaded with a transposon comprising a transposase recognition sequence and a 5’ overhang comprising the hybridization sequence.

82. The method of any of claims 80-81 , further comprising: e) amplifying and, optionally, sequencing, the isolated nucleic acid.

83. A multiplex method comprising: a) contacting chromatin with a plurality of different binding agents, wherein each of a plurality of the different binding agents binds to different targets in the chromatin, and allowing the binding agent to bind the target; b) contacting the binding agents with a plurality of different primary modification complexes, wherein each of a plurality of the different primary modification complex comprises:(I) a nucleic acid modification enzyme bound to a recruitment nucleic acid molecule comprising a different recruitment sequence;(II) an affinity agent having binding affinity for a different binding agent, and allowing the affinity agents to bind the binding agents; c) contacting the recruitment sequences with a plurality of different secondary modification complexes, wherein each of a plurality of the different secondary modification complexes comprises a nucleic acid modification enzyme bound to a hybridization nucleic acid molecule comprising a different hybridization sequence, wherein the different hybridization sequences hybridize to different recruitment sequences; and allowing the hybridization sequences to hybridize with the recruitment sequences; and d) allowing the nucleic acid modification enzyme or enzymes to modify nucleic acid in the chromatin.

84. The method of claim 83, wherein the affinity agents comprise different secondary affinity agents having affinity for the different binding agents, and an intermediate affinity element having affinity for the secondary affinity agent.

85. The method of claim 84, wherein the binding agents comprise a primary antibody, the secondary affinity agents comprise antibodies with affinity for the different primary antibodies, and the intermediate affinity elements comprise protein A or protein G bound to the primary affinity element.

86. The method of claim 85, wherein the affinity agent comprises a fusion protein comprising a transposase and protein A or protein G.

87. The method of claim 80, wherein the different first modification complexes comprise transposomes comprising first and second transposases, wherein the first transposases are loaded with transposons comprising transposase recognition sequences and a 5’ overhang comprising the different recruitment sequences.

88. The method of claim 87, wherein the different second modification complexes comprise transposomes comprising first and second transposases, wherein the firsttransposases are loaded with transposons comprising transposase recognition sequences and a 5’ overhang comprising the different recruitment sequences.

89. The method of any of claims 83-88, wherein the primary antibodies comprise antibodies of different animal species.

90. The method of any of claims 83-89, wherein the secondary binding agents comprise secondary antibodies.

91. The method of claim 90, wherein different secondary antibodies recognize antibodies of different species.

92. The method of claim 90, wherein the first complexes are bound to the secondary binding agents through protein A or protein G.

93. The method of claim 92, wherein protein A or protein G are comprised in a fusion protein with a transposase.

94. The method of any of claims 83-93, further comprising: e) amplifying and, optionally, sequencing, the isolated nucleic acid.

95. The method of any of claims 80-93, further comprising: d) isolating the modified nucleic acid; and, optionally, e) analyzing the isolated nucleic acid.

96. A method for detecting or mapping nucleic acid and nucleic acid-associated features of interest comprising: contacting said feature with a binding agent that specifically recognizes the feature of interest, wherein the binding agent is coupled to a first hybridization means; adding at least one nucleic acid modification means coupled to (i) at least one second hybridization means complementary to the first hybridization means and (ii) a nucleic acid sequencing or isolation means; allowing the first hybridization means to hybridize to the at least one second complementary hybridization means; activating the nucleic acid modification means to modify the nucleic acid proximal to the feature of interest; isolating or sequencing the modified nucleic acid using the sequencing or isolation means, thereby mapping or detecting the location in the nucleic acid of the feature of interest.

97. The method of claim 96, wherein: a) the binding agent comprises an antibody or other binding protein b) the hybridization means comprises single-strand oligonucleotides c) the nucleic acid modification means comprises a transposase d) the nucleic acid sequencing or isolation means comprises a priming adaptor98. The method of claims 96 or 97, wherein the nucleic acid modification means comprises a DNase, an MNase (which digests single-stranded DNA (ssDNA), double-strandedDNA (dsDNA), and RNA with a preference forAT-rich sequences), a DNA nicking enzyme, a biotinylating enzyme, an endonuclease (e.g., a restriction enzyme, endonuclease I), an RNAse (e.g., RNase H), a topoisomerase, a reverse transcriptase, a polyA polymerase, a terminal transferase, Cas9, Cas12, TALENs, or T7.

99. The method of claims 96-98 wherein the nucleic acid modification means coupled to the second hybridization means further comprises at least one additional hybridization means for hybridizing to and recruiting further nucleic acid modification means coupled to hybridization means complementary to said at least one additional hybridization means.

100. A complex for detecting or mapping nucleic acid and nucleic acid-associated features of interest comprising: a) a binding agent that specifically recognizes a nucleic acid or nucleic acid associated feature of interest, wherein the binding agent is coupled to a first hybridization means; b) at least one nucleic acid modification means coupled to (i) at least one second hybridization means complementary to the first hybridization means and (ii) a nucleic acid sequencing or isolation means101. A complex for detecting or mapping nucleic acid and nucleic acid-associated features of interest comprising the complex of claim 100, plus at least one additional hybridization means for hybridizing to and recruiting further nucleic acid modification means coupled to said at least one additional hybridization means.

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