ANALYSIS OF CRISPR-CAS BINDING AND CLEAVAGE SITES FOLLOWED BY HIGH-THROUGHPUT SEQUENCING (ABC-SEQ)

Abstract

The present invention relates to a method for the analysis of binding and cleavage sites followed by high-throughput sequencing. This method is called “ABC-seq”. The method is based on CUT&RUN (or CUT&Tag), originally developed for the detection of epigenetic marks, in combination with recombinant catalytically active or inactive Cas and a bioinformatics pipeline to identify off-site binding and off-site cleavage events in parallel.

Claims

1) The method of claim 16 to comprehensively capture and analyze CRISPR-Cas binding and cleavage sites, wherein step (a) comprises expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) and a single or several sgRNA in target cells, wherein the antibody of step (d) is an anti-Cas antibody, wherein the MNase of step (e) is a ProteinA-ProteinG-MNase fusion protein (pAG-MNase), wherein step (g) comprises adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, wherein step (i) comprises obtaining chromatin fragments and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant.

2) The method of claim 1, wherein in step (a) 3′ repair exonuclease 2 (Trex2) is added.

3) The method of claim 16 to comprehensively capture and analyze CRISPR-Cas binding and cleavage sites independently of an sgRNA, wherein step (a) comprises expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas protein without sgRNA, wherein the antibody of step (d) is an anti-Cas antibody, wherein step (e) comprises incubating the product of step (d) with ProteinA and/or ProteinG-MNase fusion protein (pAG-MNase), wherein step (g) comprises adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, wherein step (i) comprises pelletizing the obtained chromatin fragments and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant.

4) The method of claim 16 to validate CRISPR-Cas binding and cleavage sites, wherein step (a) comprises expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas protein containing a protein tag and an sgRNA in target cells, wherein the antibody of step (d) is an antibody against the tag of the protein of step (a), wherein step (e) comprises incubating the product of step (d) with ProteinA-MNase (pAG-MNase), wherein step (g) comprises adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, wherein step (i) comprises pelletizing the obtained oligonucleosome and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant.

5) The method of claim 16, wherein in step (f) the pAG-MNase is contained in a digitonin-containing buffer.

6) The method of claim 16 to comprehensively capture and analyze CRISPR-Cas binding and cleavage sites, wherein step (a) comprises expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) and a single or several sgRNA in target cells, wherein the antibody of step (d) is an anti-dCas antibody, wherein step (e) comprises incubating the product of step (d) with a secondary antibody against the anti-CRISPR-dCas antibody, wherein step (f) comprises incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein (pAG-Tn5) loaded with DNA primers duplexes for high-throughput sequencing, wherein step (g) comprises adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, wherein step (i) comprises pelletizing the obtained oligonucleosome and obtaining pAG-Tn5 bound digested chromatin fragments from the supernatant.

7) The method of claim 6, wherein in step (a) 3′ repair exonuclease 2 (Trex2) is added.

8) The method of claim 16 to comprehensively capture and analyze CRISPR-Cas binding and cleavage sites independently of an sgRNA, wherein step (a) comprises expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) in target cells, wherein the antibody of step (d) is an anti-dCas antibody, wherein step (e) comprises incubating the product of step (d) with a secondary antibody against the anti-CRISPR-dCas antibody, wherein step (f) comprises incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein (pAG-Tn5) loaded with DNA primers duplexes for high-throughput sequencing, wherein step (g) comprises adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, wherein step (i) comprises pelletizing the obtained oligonucleosome and obtaining pAG-Tn5 bound digested chromatin fragments from the supernatant.

9) The method of claim 16 method to validate CRISPR-Cas targeting, wherein step (a) comprises expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) containing a protein tag and a single or several sgRNA in target cells, wherein the antibody of step (d) is an antibody against the tag of the protein of step (a), wherein step (e) comprises incubating the product of step (d) with a secondary antibody against the anti-tag antibody. wherein step (f) comprises incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein loaded with DNA primers duplexes for high-throughput sequencing. wherein step (g) comprises adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, wherein step (i) comprises pelletizing the obtained oligonucleosome and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant.

10) The method of claim 16, wherein the protein is Cas9 or dCas9 or Cas12 or dCas12.

11) The method of claim 16, wherein the protein is Cas13 or dCas13.

12) The method of claim 16, wherein the optionally present hypotonic lysis step (b) is carried out in a HEPES-buffer containing spermidine.

13) The method of claim 16, wherein the magnetic beads in step (c) are Concanavalin A beads and/or the chelator in step (g) is ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA).

14) The method of claim 16, wherein the anti-Cas antibody in step (d) is a rabbit polyclonal anti-Cas9 antibody or mouse monoclonal anti-CRISPR-Cas9 antibody.

15) The method of claim 16, wherein in step (f) the transposome is contained in a digitonin-containing buffer.

16) A method comprising: (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas), (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, (d) Incubating the product of step (c) with an antibody, (e) Incubating the product of step (d) with an MNase, a secondary antibody or a transposome, (f) Optionally incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein (pAG-Tn5) loaded with DNA primers duplexes for high-throughput sequencing, (g) Adding of a Ca.sup.2+ ions-containing buffer to start digestion or tagmentation and release of chromatin complexes, (h) Adding of a chelator-containing buffer to stop the reaction of step (g), (i) Pelletizing obtained chromatin fragments or oligonucleosome and obtaining digested chromatin fragments from the supernatant, (j) Extracting of DNA and/or RNA, respectively, from the chromatin fragments of step (i), (k) High-throughput sequencing of DNA and/or RNA, respectively, (l) Identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0106] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0107] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0108] FIG. 1A-1B: ABC-seq (CUT& RUN) for CRISPR/Cas binding sites.

[0109] FIG. 2A-2B: ABC-seq (CUT& RUN) for CRISPR/Cas cleavage sites.

[0110] FIG. 3A-3B: ABC-seq (CUT& TAG) for CRISPR/Cas binding sites.

[0111] FIG. 4A-4B: ABC-seq (CUT&TAG) for CRISPR/Cas cleavage sites.

[0112] FIG. 5: Depiction of a nuclease (NUC) lobe composed of a RuvC nuclease domain and an HNH nuclease domain.

DETAILED DESCRIPTION OF THE INVENTION

[0113] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and/or” unless otherwise stated.

[0114] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps. The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof, is meant to encompass the items listed thereafter and additional items. Use of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed.

[0115] As used herein, the terms “synthetic” and “engineered” are used interchangeably and refer to the aspect of having been manipulated by the hand of man.

[0116] The terms “nucleic acid” and “nucleic acid molecule,” as used herein, refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, n which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single and/or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA/RNA hybrid, or include non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented in the 5′ to 3′ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

[0117] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent.

[0118] As used herein, “modifying” (“modify”) one or more target nucleic acid sequences refers to changing all or a portion of a (one or more) target nucleic acid sequence and includes the cleavage, introduction (insertion), replacement, and/or deletion (removal) of all or a portion of a target nucleic acid sequence. All or a portion of a target nucleic acid sequence can be completely or partially modified using the methods provided herein. For example, modifying a target nucleic acid sequence includes replacing all or a portion of a target nucleic acid sequence with one or more nucleotides (e.g., an exogenous nucleic acid sequence) or removing or deleting all or a portion (e.g., one or more nucleotides) of a target nucleic acid sequence. Modifying the one or more target nucleic acid sequences also includes introducing or inserting one or more nucleotides (e.g., an exogenous sequence) into (within) one or more target nucleic acid sequences.

[0119] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0120] The terms “Protein A-MNase”, “Protein G-MNase”, “Protein A-protein G-MNase”, “pA-MNase”, “pG-MNase”, and “pAG-Mnase” are used interchangeably herein and refer to a recombinant micrococcal nuclease-protein A, micrococcal nuclease-protein G, or micrococcal nuclease-protein A-protein G fusion protein.

[0121] The terms “Protein A-Tn5”, “Protein G-Tn5”, “Protein A-protein G-Tn5”, “pA-Tn5”, “pG-Tn5”, and “pAG-Tn5” are used interchangeably herein and refer to a recombinant hyperactive transposase 5-protein A, hyperactive transposase 5-protein G, or hyperactive transposase 5-protein A-protein G fusion protein. The term “transposome” refers to a protein A and/or protein G-Tn5 loaded with oligonucleotide duplex adapters high-throughput sequencing.

[0122] In general, a CRISPR-Cas or CRISPR system as used in herein and in documents, such as WO 2014/093622 (PCT/US2013/074667), refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a traer (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and transactivating RNA (tracrRNA) or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).

[0123] A protospacer adjacent motif (PAM) or PAM-like motif directs binding of the effector protein complex to the target locus of interest. The PAM may be a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer) or a 3′ PAM (i.e., located downstream of the 5′ end of the protospacer). The term “PAM” may be used interchangeably with the term “PFS” or “protospacer flanking site” or “protospacer flanking sequence”.

[0124] In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence may comprise DNA or RNA polynucleotides. The term “target DNA or RNA” refers to a DNA or RNA polynucleotide being or comprising the target sequence. In other words, the target RNA may be a polynucleotide or a part of a polynucleotide to which a part of the gRNA, i.e. the guide sequence, is designed to have complementarity and to which the effector function mediated by the complex comprising CRISPR effector protein and a gRNA is to be directed. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

[0125] Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas12 or Cas13. According to the present invention Class 2 subtypes II, V, and VI SpCas9, SaCas9, Cas12a/Cpf1 and Cas13 are preferred.

[0126] As described above, CRISPR-Cas based nucleic acid manipulation opens many venues for any applications ranging from basic research to gene therapy and genome engineering because of the great flexibility of the system in terms of binding specificity and functionality inherent to the CRISPR-Cas nucleoproteins themselves or endowed by possible fusion proteins (Jiang, F. & Doudna, J. A., CRISPR-Cas9 Structures and Mechanisms. Annu. Rev. Biophys. 46, 505-529 (2017); PMID 28375731). For any of these applications, reducing or entirely avoiding any unwanted off-target effects at sites with sequence homology to the targeted sites is paramount.

[0127] Engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) does already allow mapping of dCas, e.g. dCas9, binding sites. However, the readout for enChIP is either qPCR or NGS. Monitoring of enChIP enrichment of dCas9 binding sites is restricted to known binding sites due to the selection of specific PCR oligonucleotide primers. Accordingly, the outcome of the experiment is biased. NGS on the other hand does contain a considerable amount of background compared to CUT&RUN.

[0128] Combining binding of a catalytically inactive dCas with CUT&RUN or CUT&Tag allows precise mapping of CRISPR-Cas binding sites with minimal background. Unlike enChIP followed by qPCR the approach is not biases and allows a comprehensive coverage of dCas binding sites. Like enChIP followed by NGS the reduced background signal will allow mapping of genomic binding sites to which the dCas binds strongly. In addition, dCas binding following by CUT&RUN is expected to also reveal less favorable binding sites, e.g. because of nucleotide mismatches with the PAM or the protospacer itself. Sites that are bound less frequently might remain undetected using enChIP followed by NGS but are still of high interest, e.g. if they are situated in regulatory sequences of and oncogene.

[0129] Localization of the pAG-MNase or pAG-Tn5 in the vicinity of the dCas binding sites can be achieved either using an antibody specific for the dCas used. Alternatively, a dCas containing a protein tag such as a FLAG-tag can be used in conjunction with a antibody against this tag.

[0130] The inventors are aware that Cas with two inactive nuclease domains in the NUC lobe, like dCas, no longer initiates DNA-repair through insertion of DSBs but it still binds DNA or RNA specifically. A fusion of such a protein to a protein or protein domain with a particular activity allows targeted manipulation of genomic loci. Binding of catalytically inactive dCas to transcription start sites have been shown to repress transcription by blocking the transcription initiation site. This CRISPRi can also be achieved by fusing transcriptional repressor domains such as the Krüppel associated box (KRAB) domain to a dCas unit. Likewise, specific genes can be activated using CRISPRa employing dCas and transcriptional activators such as Vp64.

[0131] Base editors are fusions of a dead CRISPR-dCas and cytosine base editors (CBE) or adenine base editors (ABE). CBEs like APOBEC convert cytidine to uridine which is subsequently converted to thymidine by the cell's base excision repair (BER) mechanism. The results is a cytidine to thymidine transition and adenine to guanine respectively for the opposite DNA strand. Engineered ABEs convert adenosine to inosine, thus creating an adenine to guanine transition and thymidine to cytidine on the opposite strand. Manipulation of specific bases using base editors is generally higher than genomic modifications using HDR.

[0132] Similar to enabling CRISPRi and CRISPRa fusions of CRISPR-dCas with epigenetic modifiers like histone acyltransferases, methyltransferases, or enzymes involved in DNA de-/methylation enables targeted manipulation of epigenetic marks. It is such possible to introduce inheritable gene expression markers unlike temporary CRISPRi and CRISPRa without the need to generate DSBs.

[0133] These are just a few of the possible applications for CRISPR-Cas. Catalytically active CRISPR-Cas, nickases, or dead nucleases can be used in virtually any application relying on a site specific interaction with DNA. Because of the ease to alter DNA binding specificity by using different gRNA the system is extremely flexible. In addition, various gRNAs can be used at the same time in order to target various genomic sites simultaneously. The “multiplexing” allows e.g. editing various genomic sites at once or the deletion of larger regions by removing sequences between two gRNA target sites.

[0134] An antibody specific for the protein of interest is crucial to direct the pAG-MNase mediated nucleic acid cleavage to the intended site. The Protein A/G portion tethers the fusion protein to the Fc region of the antibody bound to its antigen. This allows the pAG-MNase nuclease portion to cleave the nucleic acid under the targeted protein and to release the nucleic acid.

[0135] Depending on the host species and isotype of the antibody and the Protein A and/or Protein G MNase fusion protein, it can be necessary to include a secondary antibody for pAG-MNase binding (Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, 1-35 (2017)). For example, if the pA-MNase is used in conjunction with a primary mouse IgG1 or goat IgG antibody, it has advantages to use a rabbit secondary antibody. Protein A binds well to rabbit or guinea pig IgG antibodies but only poorly to mouse IgG1 or goat IgG. No additional secondary antibody is needed for CUT&RUN when using pAG-MNase (Meers, M. P., Bryson, T. D., Henikoff, J. G. & Henikoff, S. Improved CUT&RUN chromatin profiling tools. Elife 8, e46314 (2019); PMID 31232687).

[0136] CUT&RUN Sets are commercially available from the company “antibodies-online GmbH”, Aachen, Germany (www.antibodies-online.com).

[0137] The CUT&RUN Positive Control (e.g. Antibodies-online GmbH, #ABIN3023255) and CUT&RUN Negative Control (e.g. Antibodies-online GmbH, #ABIN6923140) are for assessing cleavage and chromatin release without the need to sequence the released DNA fragments. It is not recommended to use a no-antibody negative control: untethered pAG-MNase will non-specifically bind and cleave any accessible DNA, thus increasing background signal.

[0138] As outlined above, both cleavage-competent and—incompetent CRISPR-Cas ribonucleoproteins have distinct applications. Depending on the application, target site binding or target-site cleavage is a requirement. CRISPR-Cas DNA binding is a much more frequent event than DNA cleavage. However, both options have to be quantitatively accounted for.

[0139] Various experimental methods exist to interrogate cleavage events indirectly by tracking changes introduced through DSB repair, e.g. CIRCLE-seq, Digenome-seq, GUIDE-seq. Other methods detect capture of defined DNA fragments in DSB sites trough linear amplification, e.g. BLISS, GUIDE-seq. These methods map CRISPR-Cas off-site cleavage events indirectly by detecting DSB repair products on the DNA level. DISCOVER-seq on the other hand tracks MRE11 recruitment sites as a correlate for DSBs: DNA bound to the MRE11 protein is enriched through ChIP, followed by high-throughput sequencing. All of these methods track cleavage events. They do not detect DNA binding events.

[0140] It should also be noted that some of these methods may pose problems which limit their applicability. GUIDE-seq for example requires delivery of chemically modified double stranded oligonucleotides which can be detrimental to certain primary cells types and complicate its use in tissue samples. Digenome-seq is carried out in vitro on isolated genomic DNA and is consequently not suitable to identify off-target binding in situ.

[0141] In contrast, the aforementioned enChIP method does detect off-site binding of endonucleases expressed with protein tags such as a FLAG-tag. Bound DNA sequences are enriched via ChIP using a tag-specific antibody. The enChIP readout was originally qPCR of putative binding sites predicted by a computer-based algorithm based on certain assumptions regarding the protein's tolerance of base pair variations within its binding site. This shortcoming can be balanced by high-throughput sequencing readout of the enriched DNA sequences. However, enChIP can only detect binding sites, not cleavage sites, and it comes with ChIP-seq inherent issues such as the necessity for a high sequencing depth and low signal-to-noise ratio

[0142] Disclosed herein is a method for detecting the binding of RGENs to genomic DNA in-situ in a cell or a population of cells. The present invention represents a major improvement compared to methods known in the art for mapping of genome wide interactions of RGENs such as CRISPR/Cas9 and other DNA modifying enzymes including but not limited to TALENs, ZFN, and meganucleases with chromatin in situ.

[0143] In certain embodiments, the interaction of the DNA modifying protein with the DNA component of chromatin is restricted to DNA binding. A catalytically inactive RGEN, e.g. CRISPR/dCas9, binds to its DNA substrate (FIG. 1 right panel and FIG. 2, right panel). Subsequently, the DNA binding sites are enriched using CUT&RUN. In short, cells are immobilized on a solid support, permeabilized using a mild detergent (e.g. digitonin), and an RGEN-specific immunoglobulin antibody binds the RGEN bound to its DNA substrate. Subsequently protein A and/or Protein G micrococcal nuclease fusion protein (pA/G-MNase) binds to the Fc fragment of the antibody via its protein A and/or protein G portion, thus tethering the MNase to the protein of interest prior to DNA fragmentation by the MNase. Upon initiation of MNase cleavage, complexes consisting of chromatin, the catalytically inactive RGEN, the antibody, and the pA/G-MNase are released and diffuse out of the cells. DNA is prepared from these complexes, converted into a sequencing library, and subjected to high-throughput sequencing. Sequencing reads are aligned to a reference genome and peaks corresponding to RGEN binding sites are identified.

[0144] In certain embodiments, the DNA modifying protein is a catalytically active RGEN; e.g. CRISPR/Cas9 (FIG. 1 left panel and FIG. 2, left panel). The RGEN binds to its DNA substrate and a subset of these bound sequences is then cleaved by the active endonuclease. Sequences that are bound but not cleaved and sequences that are cleaved and non-mutagenically repaired are identified using CUT&RUN. Sequences that are cleaved and mutagenized during the DSB repair are isolated using CUT&RUN and sequenced. Indels alter the DNA sequence so that it cannot be bound anymore by the RGEN. The corresponding sequencing reads do not align to the reference genome. Alternatively, the RGEN cleaves its DNA substrate once the DSB has been generated. Consequently, no sequencing reads are generated in this position. In both cases, peaks are missing at the positions of the DSB.

[0145] A comparison of the data generated with the catalytically inactive RGEN and the catalytically active RGEN reveals binding and cleavage sites: peaks that are present in both data sets correspond to binding sites. Peaks that are only present in the data set generated using the catalytically inactive RGEN correspond to cleavage sites. Thus, ABC-seq can detect both RGEN binding sites and cleavage sites. Methods known in the art are only suitable to detect either DNA binding sites or cleavage sites.

[0146] In other words, the present invention is based on the expression of an active and an inactive RGEN (e.g. CRISPR/Cas) with identical target sequence(s) in two parallel experiments: a binding and a binding/cleavage occurrence. The bioinformatic comparison of the identified sequencing peaks for the inactive (only binding) and the active Cas (binding and cleavage) provides a clear distinction since peaks which occur only in connection with the inactive enzyme but disappear when using the active enzyme are identified as cleavage sites.

[0147] In certain embodiments, CUT&Tag is used for the enrichment of the DNA binding sites (FIG. 3 right panel and FIG. 4, right panel). In short, cells are immobilized on a solid support, permeabilized using a mild detergent (e.g. digitonin), and an RGEN-specific immunoglobulin antibody binds the RGEN bound to its DNA substrate. A secondary antibody binds to the first antibody. Subsequently a protein A and/or Protein G hyperactive transposase 5 (pA/G-Tn5) binds to the Fc fragments of the antibodies via its protein A and/or protein G portion, thus tethering the Tn5 to the protein of interest. Upon initiation of tagmentation, the transposome attaches the sequencing adapter to the DNA ends and complexes consisting of chromatin, the catalytically inactive RGEN, the antibody, and the pA/G-Tn5 are released and diffuse out of the cells. DNA is prepared from these complexes and subjected to high-throughput sequencing. Sequencing reads are aligned to a reference genome and peaks corresponding to RGEN binding sites are identified.

[0148] In certain embodiments, genome wide DNA cleavage sites of a catalytically active RGEN; e.g. CRISPR/Cas9 are enriched using CUT&Tag (FIG. 3 left panel and FIG. 4, left panel). The RGEN binds to its DNA substrate and a subset of these bound sequences is then cleaved by the active endonuclease. Sequences that are bound but not cleaved and sequences that are cleaved and non-mutagenically repaired are identified using CUT&RUN. Sequences that are cleaved and mutagenized during the DSB repair are isolated using CUT&RUN and sequenced. Indels alter the DNA sequence so that it cannot be bound anymore by the RGEN. The corresponding sequencing reads do not align to the reference genome. Alternatively, the RGEN cleaves its DNA substrate once the DSB has been generated. Consequently, no sequencing reads are generated in this position. In both cases, peaks are missing at the positions of the DSB.

[0149] In other embodiments, isolated nuclei or tissue samples can be used instead of cells as sample material.

[0150] In certain embodiments, the 3′ repair exonuclease 2 (Trex2) is added simultaneously with the catalytically active RGEN to avoid a repeat target site cleavage and repair cycle. Trex2 has been shown to drive mutagenic DSB repair. Erroneous repair subsequently to Cas cleavage increases the number of sequencing reads in the position of a DSB that do not align with the reference genome, thus facilitating the identification of RGEN cleavage sites (Certo, M., Nature Methods 9, No. 10 (2012), pp. 973-975; PMID 22941364/US 2016/0304855 A1).

[0151] In one embodiment, the RGEN and Trex2 are delivered simultaneously by lipid transfection or electroporation. In a different embodiment, both enzymes are simultaneously expressed ectopically from recombinant expression plasmids or co-expressed from the same expression plasmid.

[0152] The following examples are intended to illustrate various embodiments of the invention. As such, they are not to be understood as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein.

[0153] Particularly, the present invention concerns a method to validate CRISPR-Cas targeting comprising the following steps: [0154] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas protein and a single or several sgRNA in target cells, [0155] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0156] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, preferably Concanvalin A beads, [0157] (d) Incubating the product of step (c) with an anti-Cas IgG antibody, preferably rabbit anti-Cas IgG antibody, [0158] (e) Optionally incubating the product of step (d) with a secondary IgG antibody, preferably anti-rabbit IgG antibody, [0159] (f) Incubating the product of step (e) with ProteinA and/or ProteinG-MNase fusion protein (pAG-MNase), [0160] (g) Adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, [0161] (h) Adding of a chelator-containing buffer to stop the reaction of step (g) [0162] (i) Pelletizing the obtained chromatin fragments and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0163] (j) Extracting of DNA and RNA, respectively, from the chromatin fragments of step (i), [0164] (k) High-throughput sequencing of DNA and RNA, respectively, [0165] (l) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).

[0166] In an alternative embodiment, the present invention concerns a method to validate CRISPR-Cas targeting comprising the following steps: [0167] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas), a single or several sgRNA, and Trex2 in target cells, [0168] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0169] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, preferably Concanvalin A beads, [0170] (d) Incubating the product of step (c) with an anti-Cas IgG antibody, preferably a rabbit anti-Cas IgG antibody, [0171] (e) Optionally incubating the product of step (d) with a secondary IgG antibody, preferably anti-rabbit IgG antibody [0172] (f) Incubating the product of step (e) with ProteinA-ProteinG-MNase fusion protein (pAG-MNase), [0173] (g) Adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, [0174] (h) Adding of a chelator-containing buffer to stop the reaction of step (g), [0175] (i) Pelletizing the obtained chromatin fragments and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0176] (j) Extracting of DNA and RNA, respectively, from the chromatin fragments of step (i), [0177] (k) High-throughput sequencing of DNA and RNA, respectively, [0178] (l) Identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).

[0179] In an alternative embodiment, the present invention concerns a method to validate CRISPR-Cas targeting comprising the following steps: [0180] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas protein without sgRNA, [0181] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0182] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, preferably Concanvalin A beads, [0183] (d) Incubating the product of step (c) with an anti-Cas IgG antibody, preferably rabbit anti-Cas IgG antibody, [0184] (e) Optionally incubating the product of step (d) with a secondary IgG antibody, preferably anti-rabbit IgG antibody, [0185] (f) Incubating the product of step (e) with ProteinA and/or ProteinG-MNase fusion protein (pAG-MNase), [0186] (g) Adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, [0187] (h) Adding of a chelator-containing buffer to stop the reaction of step (h), [0188] (i) Pelletizing the obtained chromatin fragments and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0189] (j) Extracting of DNA and RNA, respectively, from the chromatin fragments of step (i), [0190] (k) High-throughput sequencing of DNA and RNA, respectively, [0191] (l) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).

[0192] In an alternative embodiment, the present invention concerns a method to validate CRISPR-Cas targeting comprising the following steps: [0193] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas protein containing a protein tag and a sgRNA in target cells, [0194] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0195] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, preferably Concanvalin A beads [0196] (d) Incubating the product of step (c) with an IgG antibody against the tag of the protein tag of step (a), preferably a rabbit IgG antibody, [0197] (e) Optionally incubating the product of step (d) with a secondary IgG antibody, preferably an anti-rabbit IgG antibody, [0198] (f) Incubating the product of step (e) with ProteinA-MNase (pAG-MNase), [0199] (g) Adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, [0200] (h) Adding of a chelator-containing buffer to stop the reaction of step (g), [0201] (i) Pelletizing the obtained oligonucleosome and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0202] (j) Extracting of DNA and RNA, respectively, from the chromatin fragments of step (i), [0203] (k) High-throughput sequencing of DNA and RNA, respectively, [0204] (m) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).

[0205] In an alternative embodiment, the present invention concerns a method to validate CRISPR-Cas targeting comprising the following steps: [0206] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) and a single or several sgRNA in target cells, [0207] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0208] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, preferably Concanvalin A beads [0209] (d) Incubating the product of step (c) with an IgG antibody against the tag of the protein of step (a), preferably a rabbit IgG antibody, [0210] (e) Incubating the product of step (d) with a secondary IgG antibody, preferably an anti-rabbit IgG antibody [0211] (f) Incubating the product of step (e) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein (pAG-Tn5) loaded with DNA primers duplexes for high-throughput sequencing, [0212] (g) Adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, [0213] (h) Adding of a chelator-containing buffer to stop the reaction of step (g), [0214] (i) Pelletizing the obtained oligonucleosome and obtaining pAG-Tn5 bound digested chromatin fragments from the supernatant, [0215] (j) Extracting of DNA from the chromatin fragments of step (i), [0216] (k) High-throughput sequencing of DNA. [0217] (l) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).

[0218] In an alternative embodiment, the present invention concerns a method to validate CRISPR-Cas targeting comprising the following steps: [0219] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas), a single or several sgRNA, and Trex2 in target cells, [0220] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0221] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, preferably Concanvalin A beads, [0222] (d) Incubating the product of step (c) with an anti-Cas IgG antibody, preferably a rabbit anti-Cas IgG antibody [0223] (e) Incubating the product of step (d) with a secondary IgG antibody, preferably anti-rabbit IgG antibody; [0224] (f) Incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein (pAG-Tn5) loaded with DNA primers duplexes for high-throughput sequencing, [0225] (g) Adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, [0226] (h) Adding of a chelator-containing buffer to stop the reaction of step (g), [0227] (i) Pelletizing the obtained oligonucleosome and obtaining pAG-Tn5 bound digested chromatin fragments from the supernatant, [0228] (j) Extracting of DNA from the chromatin fragments of step (i), [0229] (k) High-throughput sequencing of DNA, [0230] (l) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).

[0231] In an alternative embodiment, the present invention concerns a method to validate CRISPR-Cas targeting comprising the following steps: [0232] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) containing a protein tag and a single or several sgRNA in target cells, [0233] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0234] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, preferably Concanvalin A beads [0235] (d) Incubating the product of step (c) with an IgG antibody against the tag of the protein of step (a), preferably a rabbit IgG antibody [0236] (e) Incubating the product of step (d) with a secondary IgG antibody, preferably anti-rabbit antibody [0237] (f) Incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein loaded with DNA primers duplexes for high-throughput sequencing. [0238] (g) Adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, [0239] (h) Adding of a chelator-containing buffer to stop the reaction of step (f), [0240] (i) Pelletizing the obtained oligonucleosome and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0241] (j) Extracting of DNA from the chromatin fragments of step (h), [0242] (l) High-throughput sequencing of DNA, [0243] (l) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).

[0244] MNase digestion and cleavage product release can be achieved under standard CUT&RUN conditions or high Ca.sup.2+/low salt conditions. The latter options is particularly preferable for smaller sample sizes, as it can potentially reduce background signals.

[0245] This protocol option corresponds to a more recent improvement of the CUT&RUN protocol (Meers, M. P., Bryson, T. D., Henikoff, J. G. & Henikoff, S. Improved CUT&RUN chromatin profiling tools. Elife 8, e46314 (2019); PMID 31232687). It is intended to reduce background due to DNA overdigestion by free pAG-MNase-antibody-chromatin complexes.

[0246] The protocol takes advantage of the fact that nucleosomes aggregate in the presence of high concentrations of divalent cations (e.g. 5-20 mM, preferably 7-15 mM, more preferably about 10 mM Ca.sup.2+) and at low salt concentrations (e.g. 10-50 mM, preferably about 25 mM) to reduce release of the pAG-MNase-antibody-chromatin cleavage products. Subsequently to the digestion of the samples in high Ca.sup.2+/low salt conditions, cleavage products are released in a high salt buffer containing a chelator (e.g. ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA)) to prevent further DNA cleavage.

[0247] As mentioned above, premature release of cleavage product particles during the digestion step can cause MNase off-site cleavage and thus increased background signal. This is particularly relevant when cleaving chromatin under abundant targets for longer digestions times causes increased background. Longer retention of the cleavage product particles within the nucleus could also improve CUT&RUN with lower cell numbers.

[0248] Heterologous spike-in DNA in the Stop Buffer allows the comparison of DNA yields between different samples. The total number of spike-in DNA sequencing reads serve as normalization factor and are inversely proportional to the total number of sample DNA sequencing reads (Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, 1-35 (2017); PMID 28079019). Spike-in DNA should be fragmented down to an average length of approximately 100-300 bp, preferably about 200 bp. The amount of spike-in DNA can be adjusted based on the number of cells collected for each sample: use 50-200 pg/mL, preferably about 100 pg/mL for 10.sup.4-10.sup.6 cells and 0.5-5 pg/mL, preferably about 2 pg/mL for 10.sup.2-10.sup.4 cells.

[0249] Alternatively, E. coli carry-over DNA from the purification of the pAG-MNase fusion protein has been shown to be a viable calibration standard (Meers, M. P., Bryson, T. D., Henikoff, J. G. & Henikoff, S. Improved CUT&RUN chromatin profiling tools. Elife 8, e46314 (2019); PMID 31232687). As it is introduced at step 46 in the following Example 1, it is digested by the MNase and released at the same time as the sample chromatin DNA. In this case, no heterologous spike-in DNA needs to be added to the Stop Buffer.

[0250] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

[0251] The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

EXAMPLES

1. Example 1: ABC-seq (CUT&RUN) Using High Ca.SUP.2+./Low Salt Chromatin Cleavage

[0252] 2. Buffer Preparation

[0253] Wash Buffer (100 mL)

TABLE-US-00001 Final Component Volume concentration ddH.sub.2O 94 mL — 1M HEPES pH 7.5  2 mL  20 mM 5M NaCl  3 mL 150 mM 2M Spermidine 25 μL  0.5 mM [0254] Store Wash Buffer without protease inhibitors for up to one week at 4°C. [0255] Add protease inhibitor fresh before use:

TABLE-US-00002 EDTA-free Protease Inhibitor Cocktail 100x 1 mL 1x

[0256] Binding Buffer (40 mL)

TABLE-US-00003 Final Component Volume concentration ddH.sub.2O  39 ml — 1M HEPES pH 7.5 800 μL 20 mM 1M KCl 400 μL 10 mM 1M CaCl2  40 μL  1 mM 1M MnCl2  40 μL  1 mM [0257] Store Binding Buffer for up to six months at 4° C.

[0258] Digitonin Wash Buffer (70 mL)

TABLE-US-00004 Final Component Volume concentration 5% Digitonin 350-1400 μL 0.025%-0.1% Wash Buffer 69 mL   [0259] Store Digitonin Wash Buffer for up to one day at 4° C. [0260] Recommended Digitonin concentration ranges from 0.025% to 0.1%.

[0261] Antibody Buffer (2 mL)

TABLE-US-00005 Final Component Volume concentration 0.5M EDTA 8 μL 2 mM Digitonin Wash Buffer 2 mL — [0262] Store Antibody Buffer for up to one day at 4° C. until use.

[0263] 100 mM CaCl.sub.2) (2 mL)

TABLE-US-00006 Final Component Volume concentration CaCl.sub.2   200 μL 100 mM ddH.sub.2O 1,800 μl —

[0264] Low Salt Rinse Buffer (35 mL)

TABLE-US-00007 Final Component Volume concentration ddH.sub.2O 34 mL — 1M HEPES pH 7.5  700 μL  20 mM 2M Spermidine 8.75 μL 0.5 mM 5% Digitonin  350 μL 0.05% [0265] Store Low Salt Rinse Buffer for up to one week at 4° C. until use.

[0266] Low Salt Incubation Buffer (4 mL)

TABLE-US-00008 Final Component Volume concentration ddH.sub.2O 3906 μL  — 1M HEPES pH 7.5 14 μL 3.5 mM 1M CaCl.sub.2 40 μL  10 mM 5% Digitonin 40 μL 0.05% [0267] Store Low Salt Incubation Buffer for up to one week at 4° C. until use.

[0268] Low Salt Stop Buffer (4 mL)

TABLE-US-00009 Final Component Volume concentration ddH.sub.2O 3400 μL  — 5M NaCl 136 μL 170 mM 0.2M EGTA 400 μL  20 mM [0269] Store Low Salt Stop Buffer at 4° C. until use. [0270] Add fresh before use

TABLE-US-00010 Final Component Volume concentration 5% Digitonin 40 μL 0.05% RNase A (10 mg/mL) 20 μL 50 μg/mL Glycogen (20 mg/mL)  5 μL 25 μg/mL heterologous spike-in DNA 100 pg/ml  

[0271] ABC-seq (CUT&RUN) protocol using a mouse monoclonal anti-SpCas9 antibody

I. Expression of a Catalytically Inactive Streptococcus pyogenes Cas Protein (dSpCas9) and a Catalytically Active SpCas9 in Target Cells [0272] 1. Culture human K-562 cells. [0273] 2. Transfect cells using e.g. Lipofectamine 3000 (ThermoFisher Scientific, Waltham/MA, USA) according to the manufacturer's instructions with the following expression vectors [0274] a dCas9 plasmid containing a catalytically inactive dSpCas9 construct and one or several sgRNA targeting (a) known genomic site(s) (e.g. pRP[Exp]-U6>gRNA#1-U6>gRNA#2-U6>gRNA#3-CBh>3xFlag/SV40 NLS/dCas9/NLS:T2A:EGFP/Neo, antibodies-online, Aachen, Germany) and a plasmid expressing Trex2 or [0275] a Cas9 plasmid containing a catalytically active SpCas9 construct and one or several sgRNA targeting (a) known genomic site(s) (e.g. pRP[Exp]-U6>gRNA#1-U6>gRNA#2-U6>gRNA#3-CBh>3xFlag/SV40 NLS/Cas9/NLS:T2A:EGFP/Neo, antibodies-online, Aachen, Germany) and a plasmid expressing Trex2 or [0276] a dCas9 plasmid containing a catalytically inactive dSpCas9 construct and a scrambled sgRNA (pRP[Exp]-U6>Scramble[gRNA#1]-CBh>3xFlag/SV40 NLS/dCas9/NLS:T2A:EGFP/Neo, antibodies-online, Aachen, Germany) and a plasmid expressing Trex2 or [0277] a mock transfection not containing any plasmid DNA.

II. Cell Harvest

[0278] 3. Harvest 100.000 cells for each sample at room temperature. Keep cells for each sample in separate tubes. [0279] 4. Centrifuge cell solution 3 min at 600×g at room temperature. [0280] 5. Remove the liquid carefully. [0281] 6. Gently resuspend cells in 1 mL Wash Buffer by pipetting and transfer cell solution to a 1.5 mL microcentrifuge tube. [0282] 7. Centrifuge cell solution 3 min at 600×g at room temperature and discard the supernatant. [0283] 8. Repeat steps 4-5 thrice for a total of four washes. [0284] 9. Resuspend cell pellet for each sample in 1 mL Wash Buffer by gently pipetting.

III. Concanavalin A Beads Preparation

[0285] 10. Prepare one 1.5 mL microcentrifuge tube for each sample. [0286] 11. Gently resuspend the CUT&RUN Concanavalin A Beads (antibodies-online, Aachen, Germany, #ABIN6952467). [0287] 12. Pipette 10 μL Concanavalin A Beads slurry for each sample into the 1.5 mL microcentrifuge tubes. [0288] 13. Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully. [0289] 14. Remove the microcentrifuge tube from the magnetic stand. [0290] 15. Pipette 1 mL Binding Buffer into each tube and resuspend Concanavalin A Beads by gentle pipetting. [0291] 16. Spin down the liquid from the lid with a quick pulse in a table-top centrifuge (max 100×g). [0292] 17. Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully. [0293] 18. Remove the microcentrifuge tube from the magnetic stand. [0294] 19. Repeat steps 13-16 twice for a total of three washes. [0295] 20. Gently resuspend the Concanavalin A Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 10 μL per sample.

IV. Cell Immobilization on Concanavalin A Beads

[0296] 21. Carefully vortex the cell suspension from step 9 and add 10 μL of the Concanavalin A Beads in Binding Buffer prepared in section II to each sample. [0297] 22. Close tubes tightly and rotate for 5-10 min at room temperature.

V. Cell Permeabilization and Antibody Binding

[0298] 23. Place the microcentrifuge tubes on a magnetic stand until the fluid is clear. Remove the liquid carefully. [0299] 24. Remove the microcentrifuge tubes from the magnetic stand. [0300] 25. Place each tube at a low angle on the vortex mixer set to a low speed (approximately 1,100 rpm) and add 100 μL Antibody Buffer containing digitonin. [0301] 26. Gently vortex the microcentrifuge tubes until the beads are resuspended. [0302] 27. Add 1 μL antibody corresponding to a 1:100 dilution to each tube: [0303] positive control rabbit anti-H3K4me3 antibody (antibodies-online, Aachen, Germany, #ABIN3023255) [0304] monoclonal mouse anti-CRISPR-Cas9 antibody (antibodies-online, Aachen, Germany, #ABIN4880057) [0305] polyclonal rabbit anti-CRISPR-Cas9 antibody (antibodies-online, Aachen, Germany, #ABIN5596701) [0306] negative control polyclonal guinea pig anti-rabbit IgG antibody (antibodies-online, Aachen, Germany, #ABIN101961) [0307] 28. Rotate the microcentrifuge tubes overnight at 4° C. [0308] 29. Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully. [0309] 30. Remove the microcentrifuge tubes from the magnetic stand. [0310] 31. Resuspend with 1 ml Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip. [0311] 32. Repeat steps 29-31 once for a total of two washes.

VI. Secondary Antibody Binding

[0312] 33. Place the tubes containing the samples incubated with the anti-Cas9 antibodies on a magnet stand until the fluid is clear. Remove the liquid carefully. [0313] 34. Remove the microcentrifuge tubes from the magnetic stand. [0314] 35. Vortex the sample at low speed (approx. 1,100 rpm) and add 100 μL Digitonin Wash Buffer per sample along the side of the tube. [0315] 36. Tap to remove the remaining beads from the tube side. [0316] 37. Add 1 μL of the following antibodies to a 1:100 dilution into the respective microcentrifuge tube: [0317] Polyclonal rabbit anti-mouse IgG antibody (antibodies-online, Aachen, Germany, #ABIN101785) to the tube incubated with the monoclonal mouse anti-CRISPR-Cas9 antibody (antibodies-online, Aachen, Germany, #ABIN4880057) [0318] polyclonal guinea pig anti-rabbit IgG antibody (antibodies-online, Aachen, Germany, #ABIN101961) to the tube incubated with the polyclonal rabbit anti-CRISPR-Cas9 antibody (antibodies-online, Aachen, Germany, #ABIN5596701) [0319] 38. Rotate the microcentrifuge tubes for 1 h at 4° C. [0320] 39. Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully. [0321] 40. Remove the microcentrifuge tubes from the magnetic stand. [0322] 41. Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip. [0323] 42. Repeat steps 39-41 once for a total of two washes.
VII. pAG-MNase Binding [0324] 43. Place all tubes (positive and negative control from step 32, samples from step 42) on a magnet stand until the fluid is clear. Remove the liquid carefully. [0325] 44. Remove the microcentrifuge tubes from the magnetic stand. [0326] 45. Place each tube at a low angle on the vortex mixer set to a low speed (approximately 1,100 rpm) and add 50 μL Digitonin Wash Buffer per sample alongside of the tube. [0327] 46. Add 2.5 μL pAG-MNase (antibodies-online, Aachen, Germany, #ABIN6950951). [0328] 47. Rotate the microcentrifuge tubes for 1 h at 4° C. [0329] 48. Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully. [0330] 49. Remove the microcentrifuge tubes from the magnetic stand. [0331] 50. Resuspend with 1 ml Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip. [0332] 51. Repeat steps 48-50 once for a total of two washes.
VIII. High Ca.sup.2+/Low Salt MNase Chromatin Cleavage and Release of pAG-MNase-Antibody-Chromatin Complexes. [0333] 52. Spin down the liquid from the lid with a quick pulse in a table-top centrifuge (max 100×g). [0334] 53. Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully. [0335] 54. Resuspend with 1 mL Low Salt Rinse Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 mL pipette tip. [0336] 55. Spin down the liquid from the lid with a quick pulse in a table-top centrifuge (max 100×g). [0337] 56. Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully. [0338] 57. Repeat steps 54-56 once for a total of two washes. [0339] 58. Place each tube at a low angle on the vortex mixer set to a low speed (approximately 1,100 rpm) and add 200 μL ice cold Low Salt Incubation Buffer per sample along the side of the tube. [0340] 59. Incubate tubes at 0° C. for 30 min. [0341] 60. Place the tubes on a cold magnet stand until the fluid is clear. Remove the liquid carefully. [0342] 61. Remove the microcentrifuge tubes from the magnetic stand. [0343] 62. Resuspend with 200 μL Low Salt Stop Buffer and mix by gentle vortexing. [0344] 63. Incubate tubes at 37° C. for 30 min. [0345] 64. Place the tubes on a magnet stand until the fluid is clear. [0346] 65. Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 1.5 mL microcentrifuge tubes.

IX. DNA Extraction

[0347] 66. Add 2 μL 10% SDS to a final concentration of 0.1%, 5 μL Proteinase K (10 mg/ml) to a final concentration of 2.5 mg/mL, and 1 μL RNase (10 mg/mL) to a final concentration of 50 μg/mL to each supernatant from step 65. [0348] 67. Gently vortex tubes at a low speed of approximately 1,100 rpm. [0349] 68. Incubate tubes at 50° C. for 1 h. [0350] 69. Add 200 μL PCI to tube. [0351] 70. Shake tubes thoroughly at high speed until the liquid appears milky. [0352] 71. Centrifuge tubes in a tabletop centrifuge at 16,000×g at 4° C. for 5 min. [0353] 72. Carefully transfer the upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 200 μL chloroform:isoamyl alcohol 24:1. [0354] 73. Vortex tubes thoroughly at high speed until the liquid appears milky. [0355] 74. Centrifuge tubes in a tabletop centrifuge at 16,000×g at 4° C. for 5 min. [0356] 75. Carefully transfer to upper aqueous phase to a fresh 1.5 mL microcentrifuge tube containing 2 μL glycogen (diluted 1:10 to 2 mg/mL from the 20 mg/mL stock solution). [0357] 76. Add 20 μL 3 M NaOAc pH 5.2 and 500 μL 100% ethanol. [0358] 77. Place tubes overnight at −20° C. [0359] 78. Centrifuge tubes in a tabletop centrifuge at 16,000×g at 4° C. for 20 min. [0360] 79. Remove the liquid carefully with a pipette. [0361] 80. Add 1 ml 70% ethanol. [0362] 81. Centrifuge tubes in a tabletop centrifuge at 16,000×g at 4° C. for 5 min. [0363] 82. Remove the liquid carefully with a pipette. [0364] 83. Dry the pellet in a SpeedVac. [0365] 84. Dissolve the pellet in 30 μL 1 mM Tris-HCl, 0.1 mM EDTA.

X. Sample Quality Control

[0366] 85. Determine DNA concentration using a Quantus fluorometer. [0367] 86. Check size distribution of the DNA fragments on a Tapestation

XI. Sequencing Library Preparation and Sequencing

[0368] 87. Prepare the CUT&RUN products sequencing libraries according to the workflow described in PMID 31500663 using an NEBNext Ultra II DNA Library Prep Kit for Illumina. [0369] 88. Pool sequences with different indices and perform 36 bp paired-end sequencing at a sequencing depth of 0.12× to 0.15× coverage of the human genome.
XII. Peak Calling and Comparative Analysis of SpCas and dSpCas Data Sets [0370] 89. Quality control of the sequencing reads (e.g. FastQC). [0371] 90. Trim raw sequencing reads to avoid adapter contamination in short sequencing reads. [0372] 91. Sequencing read alignment optimized for short sequencing reads (using e.g. Bowtie2). [0373] 92. Peak calling of aligned sequencing reads optimized for short sequencing reads (e.g. SEACR, MACS2). The mock transfected cells treated with the unspecific negative control antibody serves to establish a baseline. [0374] 93. Call differential peaks to identify SpCas9 binding sites and cleavage sites (e.g. Diffbind, HOMER): [0375] Peaks appearing in datasets for the catalytically inactive dSpCas9 and the active SpCas9 correspond to binding sites. [0376] Peaks appearing only in datasets for the catalytically inactive dSpCas9 but not SpCas9 correspond to cleavage sites. [0377] Peaks appearing in datasets for the catalytically inactive dSpCas9 with the specific gRNA(s) and for the catalytically inactive dSpCas9 with the scramble gRNA correspond to sequence-independent SpCas9 binding sites.

Example 2: ABC-seq (CUT&Tag)

[0378] Binding Buffer (5 mL)

TABLE-US-00011 Final Component Volume concentration ddH.sub.2O 4.85 mL — 1M HEPESpH 7.5 100 μL  20 mM 1M KCl 50 μL 10 mM 1M CaCl.sub.2  5 μL  1 mM 2.5M MnCl.sub.2 2 μl  1 mM [0379] Store Binding Buffer for up to six months at 4° C.

[0380] Wash Buffer (70 ml)

TABLE-US-00012 Final Component Volume concentration ddH.sub.2O  66 mL - 1M HEPES pH 7.5 1.4 mL  20 mM 5M NaCl 2.1 mL 150 mM [0381] Add protease inhibitor fresh before use

TABLE-US-00013 2M Spermidine 15.5 μL 0.5 mM EDTA-free Protease Inhibitor Cocktail 100×  700 μL 1× [0382] Once Spermidine and Protease Inhibitor have been added, store the Wash Buffer at 4° C. and use up within two days or store at −20° C.

[0383] Digitonin Wash Buffer (45 mL)

TABLE-US-00014 Final Component Volume concentration 5% Digitonin 225 μL 0.025% Wash Buffer 45 mL — [0384] Store Digitonin Wash Buffer for up to one day at 4° C. [0385] Recommended Digitonin concentration ranges from 0.025% to 0.1%.

TABLE-US-00015 Final Component Volume concentration 0.5M EDTA  6 μL 2 mM 10% BSA 15 μL 0.1% Digitonin Wash Buffer 1.5 mL — [0386] Store Antibody Buffer for up to one day at 4° C. until use.

[0387] Dig-300 Buffer (48 mL)

TABLE-US-00016 Final Component Volume concentration ddH.sub.2O  154 mL — 1M HEPES pH 7.5 960 μL  20 mM 5M NaCl 2.88 ml 300 mM 2M Spermidine 12 μL  0.5 mM [0388] Store Dig-300 Buffer without protease inhibitors and Digitonin for up to one week at 4° C. [0389] Add protease inhibitor and Digitonin fresh before use, e.g.

TABLE-US-00017 Protease Inhibitor Cocktail (EDTA-free) 480 μL 1× 100× 5% Digitonin  96 μL 0.01%

[0390] Tagmentation Buffer (4.2 mL)

TABLE-US-00018 Final Component Volume concentration Dig-300 Buffer 4.2 mL — 1M MgCl.sub.2 42 μl 10 mM [0391] Prepare Tagmentation Buffer fresh before use.

[0392] Oligonucleotides (for Illumina)

TABLE-US-00019 Oliognucleotide Nucleotide sequence Concentration Mosaic end- TCGTCGGCAGCGTCAGATG 100 μM adpater A TGTATAAGAGACAG (ME-A) Mosaid end- GTCTCGTGGGCTCGGAGAT 100 μM adapter B GTGTATAAGAGACAG (ME-B) Mosaic end- Phos-CTGTCTCTTATACA 100 μM reverse CATCT (ME-rev) Universal i5 AATGATACGGCGACCACCG  10 μM primer AGATCTACACTCGTCGGCA GCGTCAGATGTG Uniquely CAAGCAGAAGACGGCATAC barcoded GAGAT-8 nt barcode-  10 μM i7 primer GTCTCGTGGGCTCGGAGAT GT

[0393] ABC-Seq (CUT&Tag) Protocol Using a Mouse Monoclonal Anti-SpCas9 Antibody

I. pAG-Tn5 Adapter Complex Assembly [0394] 1. Prepare one 0.5 mL PCR tube for each of the ME-A/ME-rev and ME-B/ME-rev oligonucleotide duplexes. [0395] 2. Combine 10 μL 100 μM ME-A or ME-B oligonucleotide with 10 μM ME-rev oligonucleotide in the respective tubes. [0396] 3. Place tubes in a heating block at 95° C. for 5 min. [0397] 4. Keep tubes in the heating block and remove the heating block from the dry block incubator. Let the heating block cool down on the bench top to RT. [0398] 5. Mix 8 μl of each of the preannealed ME-A/ME-rev and ME-B/ME-rev oligonucleotide duplexes at 100 μM with 100 μL of 5.5 μM pAG-Tn5 fusion protein. [0399] 6. Incubate the mixture on a rotating platform for 1 h at RT and then store at −20° C.

II. Cell Harvest

[0400] 7. Harvest a cell number (cells as obtained in Example 1, I.) corresponding to up to 100,000 mammalian cells for the positive control, negative control, and each sample plus one at room temperature; e.g. 1.3×10.sup.6 cells for 10 samples and the two controls. [0401] 8. Centrifuge cell solution 3 min at 600×g at room temperature. [0402] 9. Remove the liquid carefully. [0403] 10. Resuspend cells in a volume of Wash Buffer corresponding to the volume of the cell solution or at most 10 mL by pipetting. [0404] 11. Centrifuge cell solution 3 min at 600×g at room temperature. [0405] 12. Remove the liquid carefully. [0406] 13. Resuspend cells in 1.2 mL Wash Buffer by pipetting and transfer cell solution to a 1.5 mL microcentrifuge tube. [0407] 14. Centrifuge cell solution 3 min at 600×g at room temperature and discard the supernatant. [0408] 15. Resuspend cell pellet in 100 μL Wash Buffer for each sample plus one by gently pipetting; e.g. 1.3 mL for 10 samples and the two controls.

III. Concanavalin A Beads Preparation

[0409] 16. Gently resuspend the CUT&RUN Concanavalin A Beads (purple dot). [0410] 17. Pipette a volume of CUT&RUN Concanavalin A Beads slurry corresponding to 10 μL for the positive control, negative control, and each sample plus one into a 1.5 mL microcentrifuge tube containing 1.2 mL Binding Buffer; e.g. 130 μL CUT&RUN Concanavalin A Beads slurry for 10 samples and the two controls. [0411] 18. Place the tube on a magnet stand until the fluid is clear. [0412] 19. Remove the liquid carefully and remove the microcentrifuge tubes from the magnetic stand. [0413] 20. Resuspend CUT&RUN Concanavalin A Beads in 1 mL Binding Buffer by gentle pipetting. [0414] 21. Spin down the liquid from the lid with a quick pulse in a table-top centrifuge (max 100×g). [0415] 22. Place the tubes on a magnet stand until the fluid is clear. [0416] 23. Remove the liquid carefully and remove the microcentrifuge tubes from the magnetic stand. [0417] 24. Repeat steps 20-23 once for a total of two washes. [0418] 25. Gently resuspend the CUT&RUN Concanavalin A Beads in a volume of Binding Buffer corresponding to the original volume of bead slurry, i.e. 10 μL per sample and control; e.g. 130 μL CUT&RUN Binding Buffer for 10 samples and the two controls.

IV. Cell Immobilization—Binding to Concanavalin A Beads

[0419] 26. Carefully vortex the cell suspension from step 15 and add the CUT&RUN Concanavalin A Beads in Binding Buffer from step 25. [0420] 27. Close tube tightly and rotate for 5-10 min at room temperature.

V. Cell Permeabilization and Primary Antibody Binding

[0421] 28. Prepare one 1.5 mL microcentrifuge tube for each sample and the two controls. [0422] 29. Place the microcentrifuge tube from step 27 on a magnetic stand until the fluid is clear. [0423] 30. Carefully remove the liquid from the cells immobilized on the CUT&RUN Concanavalin A Beads. [0424] 31. Remove the microcentrifuge tubes from the magnetic stand. [0425] 32. Gently resuspend the beads in a volume of ice cold Antibody Buffer containing digitonin corresponding to 100 μL per sample and control; e.g. 1.3 mL Antibody Buffer for 10 samples and the two controls. [0426] 33. Pipette 100 μL aliquots of the CUT&RUN Concanavalin A Beads in Antibody Buffer into the 1.5 mL microcentrifuge tubes prepared in step 28. [0427] 34. For the positive control, add 5 μL CUT&Tag rabbit anti-H3K4me3 IgG Positive Control (turquois dot) corresponding to a 1:20 dilution to the corresponding tube. [0428] 35. For the negative control, do not add anything else to the corresponding tube. [0429] 36. For the remaining samples, 1 μL anti-Cas9 primary rabbit antibody (Antibodies-online, Aachen, Germany, #ABIN2670026) corresponding to a 1:100 dilution. [0430] 37. Rotate the microcentrifuge tubes for 2 h at room temperature or overnight at 4° C. [0431] 38. Quickly spin down the liquid and place the tubes on a magnet stand until the fluid is clear. [0432] 39. Remove the liquid carefully and remove the microcentrifuge tubes from the magnetic stand.

VI. Secondary Antibody Binding

[0433] 40. Add 100 μL Digitonin Wash Buffer per tube along the side of the microcentrifuge tube and vortex at low speed (approximately 1,100 rpm). [0434] 41. Tap to remove the remaining beads from the tube side. [0435] 42. Add 5 μL CUT&Tag Secondary Antibody corresponding to a 1:20 dilution. [0436] 43. Rotate the microcentrifuge tubes for 1 h at room temperature. [0437] 44. Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. [0438] 45. Remove the liquid carefully and remove the microcentrifuge tubes from the magnetic stand. [0439] 46. Resuspend with 1 mL Digitonin Wash Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip. [0440] 47. Repeat steps 44-46 twice for a total of three washes.
VII. pAG-Tn5 Adapter Complex Binding [0441] 48. Dilute the pAG-Tn5 adapter complex ABIN from step 6 1:250 in a volume of Dig-300 Buffer corresponding to 100 μL per sample; e.g. 5.2 μL pAG-Tn5 adapter complex in 1.3 mL for for 10 samples and the two controls. [0442] 49. Place the tubes from step 47 on a magnet stand until the fluid is clear. [0443] 50. Remove the liquid carefully and remove the microcentrifuge tubes from the magnetic stand. [0444] 51. Place each tube at a low angle on the vortex mixer set to a low speed (approximately 1,100 rpm) and add 100 μL pAG-Tn5 adapter complex in Dig-300 Buffer from step 48 along the side of the tube. [0445] 52. Rotate the microcentrifuge tubes for 1 h at room temperature. [0446] 53. Spin down the liquid and place the tubes on a magnet stand until the fluid is clear. [0447] 54. Remove the liquid carefully and remove the microcentrifuge tubes from the magnetic stand. [0448] 55. Resuspend with 1 ml Dig-300 Buffer and mix by inversion. If clumping occurs, gently remove the clumps with a 1 ml pipette tip. [0449] 56. Repeat steps 53-55 twice for a total of three washes.

VIII. Tagmentation

[0450] 57. Spin down the liquid from the lid with a quick pulse in a table-top centrifuge (max 100×g). [0451] 58. Place the tubes on a magnet stand until the fluid is clear. [0452] 59. Remove the liquid carefully and remove the microcentrifuge tubes from the magnetic stand. [0453] 60. Place each tube at a low angle on the vortex mixer set to a low speed (approximately 1,100 rpm) and add 300 μL Tagmentation Buffer along the side of the tube. [0454] 61. Spin down the liquid from the lid with a quick pulse in a table-top centrifuge (max 100×g) [0455] 62. Rotate the microcentrifuge tubes for 1 h at 37° C.

IX. DNA Extraction

[0456] 63. Add 10 μL 0.5 M EDTA to a final concentration of 16 mM, 3 μL 10% SDS to a final concentration of 0.1%, and 7.5 μL Proteinase K (10 mg/mL) to a final concentration of 0.25 mg/mL to each reaction. [0457] 64. Vortex tubes thoroughly at a high speed. [0458] 65. Incubate tubes at 50° C. for 1 h or at 37° C. ON. [0459] 66. Without separating the liquid supernatant and the beads add 300 μL PCI to each tube. [0460] 67. Vortex tubes thoroughly at high speed until the liquid appears milky. [0461] 68. Transfer liquid to a 1.5 mL phase-lock tube. [0462] 69. Add 300 μL chloroform and mix by inversion. [0463] 70. Centrifuge tubes in a tabletop centrifuge at 16,000×g at room temperature for 3 min. [0464] 71. Using a pipette, transfer the aqueous layer to a new tube containing 750 μL 100% ethanol. [0465] 72. Transfer tubes to a cold tabletop centrifuge and centrifuge at 16,000×g at 4° C. for 15 min. [0466] 73. Carefully pour off the liquid and remove the remaining liquid with a pipette. [0467] 74. Add 1 mL 100% ethanol. [0468] 75. Carefully pour off the liquid, remove the remaining liquid with a pipette, and air dry the tubes. [0469] 76. Dissolve the pellet in 23 μL TE containing RNase A diluted 1:400 to 25 ng/mL. [0470] 77. Incubate tubes at 37° C. for 10 min.

X. PCR Amplification and Clean-Up

[0471] 78. Transfer 21 μl into a 0.5 mL PCR tube. [0472] 79. Add 2 μL Universal i5 Primer at 10 μM and 2 μL i7 Primer at 10 μM with a unique barcode for each sample. [0473] 80. Add 25 μL PCR master mix of a high fidelity polymerase (e.g. NEBNext Ultra II Q5 Master Mix, Roche KAPA Library Amplification Kit). [0474] 81. Mix tubes thoroughly by vortexing. [0475] 82. Spin down the liquid from the lid with a quick pulse (max 100×g). [0476] 83. PCR program:

TABLE-US-00020 step 1 58° C. 5 min step 2 72° C. 30 sec step 3 98° C. 30 sec step 4 98° C. 10 sec step 5 60° C. 10 sec step 6 goto step 4 14 times step 7 72° C. 1 min  4° C. hold [0477] 84. Transfer the PCR reactions to 1.5 mL microcentrifuge tubes. [0478] 85. Add 1.3× volumes (65 μL for a 50 μL PCR mix) SPRI bead slurry and mix by pipetting. [0479] 86. Place the tubes on a magnet stand until the fluid is clear. [0480] 87. Remove the liquid carefully with a pipette and keep the microcentrifuge tubes on the magnetic stand. [0481] 88. Add 200 μL 80% ethanol. [0482] 89. Remove the liquid carefully with a pipette and remove the microcentrifuge tubes from the magnetic stand. [0483] 90. Immediately add 25 μL 10 mM Tris-HCl pH 8.0 and mix by pipetting. Elute DNA for at RT for 5 min. [0484] 91. Place the tubes on a magnet stand until the fluid is clear. [0485] 92. Transfer liquid to fresh 1.5 mL microcentrifuge tubes.

XI. Sample Quality Control

[0486] 93. Determine DNA concentration using a Quantus fluorometer. [0487] 94. Check size distribution of the DNA fragments on a Tapestation.

XIII. Sequencing Library Preparation and Sequencing

[0488] 95. Prepare the CUT&RUN products sequencing libraries according to the workflow described in PMID 31500663 using an NEBNext Ultra II DNA Library Prep Kit for Illumine. [0489] 96. Pool sequences with different indices and perform 36 bp paired-end sequencing at a sequencing depth of 0.12× to 0.15× coverage of the human genome.
XIV. Peak Calling and Comparative Analysis of SpCas and dSpCas Data Sets [0490] 94. Quality control of the sequencing reads (e.g. FastQC). [0491] 95. Trim raw sequencing reads to avoid adapter contamination in short sequencing reads. [0492] 96. Sequencing read alignment optimized for short sequencing reads (using e.g. Bowtie2) [0493] 97. Peak calling of aligned sequencing reads optimized for short sequencing reads (e.g. SEACR, MACS2). The mock transfected cells treated with the unspecific negative control antibody serves to establish a baseline. [0494] 98. Call differential peaks to identify SpCas9 binding sites and cleavage sites (e.g. DESeq2, Diffbind, HOMER): [0495] Peaks appearing in datasets for the catalytically inactive dSpCas9 and the active SpCas9 correspond to binding sites. [0496] Peaks appearing only in datasets for the catalytically inactive dSpCas9 but not SpCas9 correspond to cleavage sites. [0497] Peaks appearing in datasets for the catalytically inactive dSpCas9 with the specific gRNA(s) and for the catalytically inactive dSpCas9 with the scramble gRNA correspond to sequence-independent SpCas9 binding sites.

[0498] The invention is further described by the following numbered paragraphs:

1) A method to comprehensively capture and analyze CRISPR-Cas binding and cleavage sites comprising the following steps:

[0499] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) and a single or several sgRNA in target cells, [0500] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0501] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, [0502] (d) Incubating the product of step (c) with an anti-Cas antibody, [0503] (e) Incubating the product of step (d) with ProteinA-ProteinG-MNase fusion protein (pAG-MNase), [0504] (f) Adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, [0505] (g) Adding of a chelator-containing buffer to stop the reaction of step (f), [0506] (h) Pelletizing the obtained chromatin fragments and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0507] (i) Extracting of DNA and RNA, respectively, from the chromatin fragments of step (h), [0508] (j) High-throughput sequencing of DNA and RNA, respectively. [0509] (k) Identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas)
2) The method of numbered paragraph 1, wherein in step (a) 3′ repair exonuclease 2 (Trex2) is added.
3) A method to comprehensively capture and analyze CRISPR-Cas binding and cleavage sites independently of an sgRNA comprising the following steps: [0510] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas protein without sgRNA, [0511] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0512] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, [0513] (d) Incubating the product of step (c) with an anti-Cas antibody, [0514] (e) Incubating the product of step (d) with ProteinA and/or ProteinG-MNase fusion protein (pAG-MNase), [0515] (f) Adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, [0516] (g) Adding of a chelator-containing buffer to stop the reaction of step (f), [0517] (h) Pelletizing the obtained chromatin fragments and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0518] (i) Extracting of DNA and RNA, respectively, from the chromatin fragments of step (h), [0519] (j) High-throughput sequencing of DNA and RNA, respectively, [0520] (k) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas)
4) A method to validate CRISPR-Cas binding and cleavage sites comprising the following steps: [0521] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas protein containing a protein tag and an sgRNA in target cells, [0522] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0523] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, [0524] (d) Incubating the product of step (c) with an antibody against the tag of the protein of step (a), [0525] (e) Incubating the product of step (d) with ProteinA-MNase (pAG-MNase), [0526] (f) Adding of a Ca.sup.2+ ions-containing buffer to start MNase digestion and release of pAG-MNase-antibody-chromatin complexes, [0527] (g) Adding of a chelator-containing buffer to stop the reaction of step (f), [0528] (h) Pelletizing the obtained oligonucleosome and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0529] (i) Extracting of DNA and RNA, respectively, from the chromatin fragments of step (h), [0530] (j) High-throughput sequencing of DNA and RNA, respectively, [0531] (k) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas)
5) The method of numbered paragraph 1, 2, 3, or 4, wherein in step (e) the pAG-MNase is contained in a digitonin-containing buffer.
6) A method to comprehensively capture and analyze CRISPR-Cas binding and cleavage sites comprising the following steps [0532] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) and a single or several sgRNA in target cells, [0533] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0534] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, [0535] (d) Incubating the product of step (c) with an anti-dCas antibody, [0536] (e) Incubating the product of step (d) with a secondary antibody against the the anti-CRISPR-dCas antibody, [0537] (f) Incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein (pAG-Tn5) loaded with DNA primers duplexes for high-throughput sequencing, [0538] (g) Adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, [0539] (h) Adding of a chelator-containing buffer to stop the reaction of step (g), [0540] (i) Pelletizing the obtained oligonucleosome and obtaining pAG-Tn5 bound digested chromatin fragments from the supernatant, [0541] (j) Extracting of DNA from the chromatin fragments of step (i), [0542] (k) High-throughput sequencing of DNA, [0543] (l) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).
7) The method of numbered paragraph 6, wherein in step (a) 3′ repair exonuclease 2 (Trex2) is added.
8) A method to comprehensively capture and analyze CRISPR-Cas binding and cleavage sites independently of an sgRNA comprising the following steps: [0544] (a) Expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) in target cells, [0545] (b) Optionally hypotonic lysis of the cells of step (a) to release nuclei, [0546] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, [0547] (d) Incubating the product of step (c) with an anti-dCas antibody, [0548] (e) Incubating the product of step (d) with a secondary antibody against the the anti-CRISPR-dCas antibody, [0549] (f) Incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein (pAG-Tn5) loaded with DNA primers duplexes for high-throughput sequencing, [0550] (g) Adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, [0551] (h) Adding of a chelator-containing buffer to stop the reaction of step (g), [0552] (i) Pelletizing the obtained oligonucleosome and obtaining pAG-Tn5 bound digested chromatin fragments from the supernatant, [0553] (j) Extracting of DNA from the chromatin fragments of step (i), [0554] (k) High-throughput sequencing of DNA, [0555] (l) Bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas)
9) A method to validate CRISPR-Cas targeting comprising the following steps: [0556] (a) expressing a catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas) containing a protein tag and a single or several sgRNA in target cells, [0557] (b) optionally hypotonic lysis of the cells of step (a) to release nuclei, [0558] (c) Immobilizing whole cells of step (a) or nuclei of step (b) with magnetic beads, [0559] (d) Incubating the product of step (c) with an antibody against the tag of the protein of step (a), [0560] (e) Incubating the product of step (d) with a secondary antibody against the the anti-tag antibody. [0561] (f) Incubating the product of step (d) with a transposome comprising a protein A and/or protein G hyperactive Tn5 fusion protein loaded with DNA primers duplexes for high-throughput sequencing. [0562] (g) Adding of a Ca.sup.2+ ions-containing buffer to start tagmentation and release of pAG-Tn5-chromatin complexes, [0563] (h) Adding of a chelator-containing buffer to stop the reaction of step (f), [0564] (i) Pelletizing the obtained oligonucleosome and obtaining pAG-MNase-bound digested chromatin fragments from the supernatant, [0565] (j) Extracting of DNA from the chromatin fragments of step (h), [0566] (l) High-throughput sequencing of DNA, [0567] (l) bioinformatic identification of differential peaks of sequencing reads in samples prepared using the catalytically inactive Cas protein (dCas) or catalytically active Cas proteins (Cas).
10) The method of any of numbered paragraphs 1 to 9, wherein the protein is Cas9 or dCas9 or Cas12 or dCas12.
11) The method of any of numbered paragraphs 1 to 5, wherein the protein is Cas13 or dCas13.
12) The method of any of numbered paragraphs 1 to 11, wherein the optionally present hypotonic lysis step (b) is carried out in a HEPES-buffer containing spermidine.
13) The method of any of numbered paragraphs 1 to 12, wherein the magnetic beads in step (c) are Concanavalin A beads and/or the chelator in step (g) is ethyleneglycol-bis(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA).
14) The method of any of numbered paragraphs 1, 2, 3, 5, 6, 7, 8, 10, 12, or 13 wherein the anti-Cas antibody in step (d) is a rabbit polyclonal anti-Cas9 antibody or mouse monoclonal anti-CRISPR-Cas9 antibody.
15) The method of numbered paragraph 6, 7, 8, 9, 10, 12, 13, or 14 wherein in step (f) the transposome is contained in a digitonin-containing buffer.

[0568] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.