It is difficult to selectively isolate desired DNA sequences from complex mixtures of double-stranded DNA (dsDNA) fragments. Techniques for selective isolation of DNA fragments of interest are typically based on DNA-oligonucleotide hybridization but protocols are long, complicated, and require high technical skills. A new technique for enrichment or depletion of DNA sequences of interest from mixtures of double-stranded DNA (dsDNA) is desirable.

The invention uses an in vitro assembled, catalytically inactive, programmable nuclease to recognize and bind those DNA fragments that contain sequences recognized by that nuclease. One example of such an in vitro assembled, catalytically inactive,

programmable nuclease is ternary ribonucleoprotein complex Cas9/crRNA tracRNA. An artificially engineered nuclease with customized target dsDNA sequence specificity may also be used. Examples of such programmable enzymes include engineered homing

endonucleases (l-Crel, l-Scel), triple helix forming oligonucleotide (TFO)-linked nucleases (Eisenschmidt et al., Developing a programmed restriction endonuclease for highly specific DNA cleavage, Nucleic Acids Research, 33 (2005) 7039, WO 2006/027099), zinc-finger nucleases ((ZFNs) (Durai et al. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells, Nucleic Acids Research, 33 (2005) 5978), or transcription-activator like effector nucleases ((TALENs) (Li et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and Fokl DNA-cleavage domain, Nucleic Acids Research 39 (201 1 ) 359).

DNA fragments bound by such catalytically inactive programmable site specific nucleases are easily isolated using the affinity tag that may be located either on the nuclease protein molecule, or in the case of Cas9 complexes on crRNA, or tracrRNA, or in the case of TFO complexes on the guiding oligonucleotide. Such specific DNA fragments isolated according to the inventive method may be used in any downstream application including, but not limited to, targeted sequencing by any of existing next-generation sequencing techniques. The method may be used for removal of undesired DNA sequences, such as those encoding ribosomal RNA in RNA sequencing experiments.

The polymerase chain reaction (PCR) was the first technique used for selective analysis of DNA regions of interest. Individual genome regions of interest were amplified using pairs of unique primers complimentary to those regions, and amplified DNA fragments were analyzed using various techniques including Sanger sequencing. Development of techniques for parallel analysis of millions of different DNA fragments of interest, the most powerful among them being microarrays and next-generation sequencing platforms, created a need for more powerful enrichment techniques. With the advent of techniques for solid surface massive parallel synthesis, the first approaches developed to capture DNA regions of interest explored oligonucleotides located on microarrays (Hodges et al., Genome-wide in situ exon capture for selective re-sequencing. Nat Genet. 39 (2007) 1522; Albert et al., Direct selection of human genomic loci by microarray hybridization. Nat Methods. 4 (2007) 903). These approaches demonstrated that high-density microarrays targeting more than 200,000 protein-coding exons may recover up to 98% of intended exons. However, these approached required using expensive instruments for manufacturing microarrays and involved complicated washing and elution steps, making the technology not amenable for high-throughput applications. This drawback was resolved by implementation of in-solution hybridization, followed by capturing biotin-labeled oligonucleotides hybridized to complimentary DNA regions on streptavidin-coated paramagnetic beads, and their sequencing (Bainbridge et al., Whole exome capture in solution with 3 Gbp of data. Genome Biol.1 1 (2010)). The solution-based method for targeted DNA capture-sequencing of the complete human exome allows discovery of greater than 95% of all expected heterozygous single base variants, requires as little as 3 Gbp of raw sequence data, and is less expensive and more amenable for high-throughput applications. Despite the fact that the aforementioned approach is less sophisticated compared to enrichment using high- density microarrays, it still requires high technical skills, and the protocol is extremely long, taking a few days. As an example, a kit from Roche for exome enrichment requires 64-72 hours for the hybridization step (SeqCap EZ Library SR User's Guide, Version 4.1 ), while preparing both the library of fragments and biotin-labeled hybridization oligonucleotides involves multiple pipetting and clean-up steps. The same is true for steps which follow the hybridization step, making the overall protocol extremely complicated and not robust. In addition, isolated DNA fragments are single-stranded, thus requiring addition of adapters prior to enrichment. Collectively, techniques developed for enrichment purposes have many drawbacks.

A methods that resolves issues specific for oligonucleotide-directed hybridization- based techniques is desirable.

Components of Type II CRISPR-Cas systems, or other site specific nucleases, that may be engineered to recognize different DNA sequences, could serve the same function and likely overcome obstacles specific for hybridization-based techniques.

Clustered, regularly interspaced, short palindromic repeats (CRISPR) / CRISPR Associated (Cas) Systems evolved as bacterial and archaeal adaptive defense systems directed towards invading phages and plasmids (Terns and Terns, CRISPR-based adaptive immune systems. Curr Opin Microbiol. 14 (201 1 ) 321 ; Bhaya et al., CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet. 45 (201 1 ) 273). These defense systems include two major components: enzymes encoded by genes of Cas operon, and small RNAs which are transcribed from the CRISPR region into a long transcript and then processed into a set of individual short CRISPR RNAs

(crRNAs). Each crRNA has bipartite structure: one is a repeat that is the same in all crRNAs of a particular CRISPR/Cas system, the other, protospacer, is a variable spacer sequence complementary to the invading nucleic acid. Bacteria harboring functionally active CRISPR- Cas systems can incorporate short sequences of invading elements within the CRISPR loci. crRNAs produced from CRISPR loci form complexes with Cas protein(s) and guide them to recognize and inactivate those invading elements that have sequences complementary to crRNAs, thus providing a "memory" for bacteria or archaea to recognize and inactivate phages and plasmids already encountered. Based on structural and functional peculiarities of CRISPR-Cas systems, they are divided into three major types (Makarova et al., Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol. 9 (201 1 ) 467). In Types I and III crRNAs and Cas proteins are the only components required for expression and maturation of crRNAs, while processing of crRNAs in Type II CRISPR-Cas systems requires the presence of one additional short RNA of fixed size, termed trans-activating crRNA (tracrRNA), which is encoded in the vicinity of the cas genes and CRISPR region (Deltcheva et al., CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 471 (201 1 ) 602). Genetic in vivo interference studies revealed that, in the case of the Streptococcus thermophilus CRISPR3-Cas system which belongs to Type II, the cas9 gene coding for Cas9 enzyme is the only gene required for CRISPR-encoded interference (Sapranauskas et al., The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.

Nucleic Acids Res. 39 (201 1 ) 9275). It was demonstrated that in vivo formed Cas9-crRNA ribonucleoprotein complex may be isolated from bacterial cells by using the protein purification tag added to the Cas9, and the isolated complex retained its ability to recognize and introduce double-stranded breaks into those DNA fragments which contained sequences complementary to the crRNA (Gasiunas et al., Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109 (2012) E2579-86). It was shown that cleavage in Streptococcus thermophilus CRISPR3-Cas system is executed by two active sites of Cas9, each introducing a nick into predetermined DNA strand at a specific position. Electrophoretic Mobility Shift Assays (EMSA) demonstrated that the isolated wild-type Cas9-crRNA complex in the absence of magnesium ions binds dsDNA fragment bearing a nucleotide sequence complementary to the protospacer. Magnesium ions are required for DNA cleavage, thus their absence in the binding reaction mixture prevents bound DNA from cleavage. Binding was efficient only if the target dsDNA included not only a sequence complementary to the protospacer of crRNA, but also had the so-called conserved

protospacer-adjacent motif (PAM) sequence NGGNG. It was also shown that the in vitro assembled Cas9-crRNA-tracRNA complex cleaves DNA, and a minimal tracrRNA region was identified that was required to accomplish its function (Karvelis et al., crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biol. 10 (2013) 841 ). Similar results were obtained by analyzing features of the other Type II CRISPR-Cas found in Streptococcus pyogenes (Jinek et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337 (2012) 816). The latter differs from Streptococcus thermophilus CRISPR3-Cas by PAM sequence which is shorter (NGG).

Catalytically active complexes include crRNA, tracrRNA and Cas9, and it was concluded that the PAM sequence is obligatory for binding of dsDNA targets. Using EMSA demonstrated that adding tracrRNA enhances target DNA binding by catalytically inactive Cas9-crRNA substantially, providing evidence that tracrRNA is required for target DNA recognition. The role of tracrRNA was speculated to orient crRNA properly for interaction with the

complementary strand of target DNA. The importance of individual regions of both crRNA and tracrRNA were analyzed, and it was found that only some of them are essential for DNA binding and/or cleavage. Based on crRNA and tracrRNA shortening results, a chimeric RNA molecule was developed; it included structural components of both tracrRNA and crRNA, and was able to program Cas9 for site-specific cleavage. The ability to program the in vivo specificity of Cas9 by tracrRNA crRNA, and especially by a single transcript, to target and cleave any dsDNA sequence of interest provided opportunities for editing and manipulations of large genomes, and much progress was made in that direction (Richter et al., Exploiting CRISPR/Cas: interference mechanisms and applications. Int J Mol Sci. 14 (2013) 14518).

Recent efforts concentrated on elucidation of CRISPR-Cas biochemical and genetic mechanisms and development of CRISPR-Cas based efficient and robust tools for editing various genomes. No attention was paid about possible alternative uses of these unique systems. The inventors realized that the ability of Cas9 enzymes, belonging to Type II CRISPR-Cas systems, to bind specific dsDNA targets in vitro depending on the sequence present on a guiding crRNA, and in the presence of tracrRNA, could be used in other important applications, including but not limited to in vitro enrichment of individual and multiple dsDNA regions of interest from complex mixtures of dsDNA fragments, as well as in vitro removal of undesired dsDNA sequences from libraries of dsDNA fragments.

The use of in vivo expressed Cas9 for isolation of a single genomic DNA locus of interest was described (Fujita and Fujii, Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Biochem Biophys Res Commun. 1 1 (2013)). The workflow was described as follows: (i) introduction of DNA or RNA coding for both the catalytically inactive Cas9 and the guide RNA into cells of interest, (ii) co-expression of both Cas9 and guide RNA to form complexes in vivo between expressed Cas9/guide RNA and genomic DNA region of interest, (iii) cross-linking macromolecules by treatment with formaldehyde, (iv) isolation of all Cas9 molecules, including those complexed with the target DNA, using immunoprecipitation using antibody against the tag added to the Cas9 mutant protein, and (v) analysis of all proteins which were co-purified with Cas9 by mass-spectrometry, attempting to identify those cellular proteins which were in close vicinity to the Cas9 presumably bound to the genomic DNA at a specific genome location.

SUMMARY

The invention describes new technique of selective isolation of double-stranded DNA fragments of interest. The technique has three major steps. In the first step there is in vitro formation of complexes between Cas9, tracrRNA and one, few, or as many as necessary different crRNA molecules which predetermine the specificity of the formed

Cas9/tracrRNA crRNA complexes towards sequence targets located within the double stranded DNA. In the second step double-stranded DNA under investigation is mixed with preformed complexes for the time required for formation of higher-order complexes between

Cas9/tracrRNA/crRNA and DNA fragments which contain nucleotide sequences

complementary to the crRNA. In the third step DNA fragments bound by Cas9/crRNA tracRNA are isolated by using the afinity tag-solid particle interaction. Affinity tags that may be used are known in the art, such as a biotin moiety, which may be located predominantly in synthetic tracrRNA, but potentially could be located in crRNA, in hybrid tracrRNA crRNA molecule, or on the surface of Cas9 protein, including polyhistidine tag, MBP maltose binding protein (MBP) tag, chitin binding domain (CBD) tag, or strepavidin tag fused to Cas9, or an antibody raised against Cas9 protein. Complexes bound by streptavidin-bearing particles are then purified from the mixture of DNA fragments and released from particles. Double-stranded DNA isolated thereby from released complexes may be used in any downstream application including, but not limited to, targeted sequencing by any of existing next-generation sequencing techniques.

The inventive method may be used for removal of undesired DNA sequences, such as those which code for ribosomal RNA in RNA sequencing experiments.

In one embodiment, the dsDNA Cas9/crRNA tracRNA complexes are isolated from the reaction mixture using the affinity tag incorporated into the structure of any of Cas9, crRNA, and/or tracRNA of the ternary ribonucleoprotein complex before its in vitro assembly. In one embodiment desired DNA sequences were enriched from mixtures of dsDNA fragments. In one embodiment the method depletes undesired DNA sequences from mixtures of dsDNA fragments.

Those skilled in the art will recognize that the inventive method can be applied to other members of Type II CRISPR-Cas systems, or other catalytically inactive programmable nucleases. Those skilled in the art will recognize that catalytic mutants of Cas9, or other programmable nucleases may be used. Catalytically inactive mutants of Cas9 useful in practicing this invention may be generated by introducing D31A mutation in the RuvC active center, or N891A mutation in the HNH domain in S. thermophilus Cas9, (Gasiunas et al., Cas9- crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl.Acad. Sci. USA 109 (2012) E2579-E2586), or in the case of S. pyogenes Cas9 by introducing D10A mutation in the RuvC active center, or H840A mutation in the HNH domain (Jinek et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337 (2012) 816), each of which is incorporated by reference herein in its entirety.

Those skilled in the art will recognize that different tags used for protein purification besides biotin can be used.

Thus, in a first aspect, the present invention relates to a method for selective in vitro isolation of double-stranded DNA, the method comprising (a) contacting a sample, preferably a biological sample, containing double-stranded DNA (dsDNA) with a catalytically inactive nuclease, where the catalytically inactive nuclease comprises a target dsDNA sequence binding specificity, preferably a customizable target dsDNA sequence binding specificity, and optionally an affinity tag, (b) incubating the catalytically inactive nuclease with the sample under conditions sufficient for forming a complex comprising the catalytically inactive nuclease and a target (or selected) dsDNA, and (c) isolating the complex from the sample. In a preferred embodiment, the sample containing dsDNA is contacted with a catalytically inactive nuclease in a solution. A solution can be, but is not limited to, a solution as used in the Examples described herein. The term -biological sample" as used herein refers to a sample obtained from a biological subject, including sample of biological tissue or fluid origin obtained in vivo or in vitro. Such samples can be, but are not limited to, body fluid (e.g., blood, blood plasma, serum, or urine), organs, tissues, fractions, cells isolated from mammals including, humans and cell organelles. Biological samples can further be, but are not limited to, fragmented dsDNA, for example fragmented dsDNA plasmids or vectors, for instance by cleavage with one or more restriction enzymes. Biological samples also may include sections of the biological sample including tissues (e.g., sectional portions of an organ or tissue). Biological samples may also include extracts from a biological sample. Biological samples may further comprise proteins, carbohydrates or nucleic acids. A biological sample may be of prokaryotic origin, archaeal origin, or eukaryotic origin (e.g., insects, protozoa, birds, fish, and reptiles). In some embodiments, the biological sample is mammalian (e.g., rat. mouse, cow, dog, donkey, guinea pig, or rabbit). In certain embodiments, the biological sample is of primate origin (e.g., example, chimpanzee, or human). The term "double-stranded DNA" or "dsDNA" as used herein refers to a deoxyribonucleotide polymer (DNA strand) hybridized to its complement through Watson- Crick bonding. The dsDNA can be of any length and can be associated with additional components (e.g., histone proteins or proteins involved in replication or transcription). One of skill will appreciate that the two strands of DNA may not be 100% complementary, so long as the percentage is high enough in the given conditions for the two strands to remain associated. The words "complementary" or "complementarity" refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. The term "catalytically inactive nuclease comprising customizable target dsDNA sequence binding specificity" as used herein refers to a catalytically inactive nuclease for which the target dsDNA sequence binding specificity can be customized, such as for example demonstrated in the examples, but not limited thereto. The term "conditions sufficient for forming a complex" as used herein refers to the conditions, such as for example, but not limited to, temperature, incubation time, buffers, that are sufficient to allow formation of a complex comprising the catalytically inactive nuclease and the target (or selected) dsDNA.

In a preferred embodiment the catalytically inactive nuclease is selected from the group consisting of a Type II CRISPR-Cas system, a homing nuclease, a triple helix forming oligonucleotide (TFO)-linked nuclease, a zinc-finger nuclease, a transcription-activator like effector nuclease (TALEN), and combinations thereof. Preferably, the Type II CRISPR-Cas system is ribonucleoprotein complex Cas9/crRNA tracRNA and prior to step (a), forming a complex in vitro between Cas9, a synthetic tracrRNA and at least one synthetic crRNA molecule that predetermines the specificity of the formed Cas9/tracrRNA crRNA complex towards a sequence target located within the double stranded DNA. Preferably, the Type II CRISPR-Cas system, more preferably the Cas9/crRNA/tracrRNA, is obtained from or modified from Streptococcus thermophilius and/or Streptococcus pyogenes.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the homing nuclease is either l-Crel or l-Scel.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the complex is isolated from the sample using the affinity tag. Preferably, the affinity tag is selected from the group consisting of biotin, desthiobiotin, streptavidin, polyhistidine, maltose binding protein (MBP), chitin binding domain (CBD), and combinations thereof.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the complex is isolated using an antibody having affinity for the catalytically inactive nuclease, or a portion thereof.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the affinity tag is attached to the Cas9 protein, the synthetic tracrRNA, the synthetic crRNA, and/or a tracrRNA crRNA hybrid duplex molecule.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment in step (c) of the method of the invention the complex is isolated from the sample by an interaction between the affinity tag and a binding molecule, the binding molecule capable of binding with the affinity tag, and the binding molecule attached to a solid support. In a preferred embodiment, the method further comprises releasing the bound complex from the solid support.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the isolated, selected dsDNA is used in targeted sequencing.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment following isolation and removal of the selected dsDNA from the sample, the sample substantially depleted of the selected dsDNA is used in downstream applications.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment

the nuclease is rendered catalytically inactive by at least one of: (a) at least one mutation in the nuclease or, when the nuclease is a complex, at least one component of the complex, where the mutation at least substantially abolishes catalytic activity; or (b) the solution is at least substantially devoid of an agent required for catalytic activity of the nuclease, or (c) a catalytic inhibitor is present. Preferably, the solution lacks or substantially lacks Mg ²⁺ .

Alternatively or in combination with any previous embodiments, in a preferred embodiment the catalytically inactive nuclease is Cas9/crRNA tracRNA and the Cas9 protein is mutated in a RuvC active center or a HNH domain to render the nuclease catalytically inactive. For example, but not limiting the present invention, nucleases that are rendered catalytically inactive are provided in the Examples. Alternatively or in combination with any previous embodiments, the dsDNA is a collection of fragmented dsDNA.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the catalytically inactive nuclease or catalytically inactive ribonucleoprotein retains binding affinity for the selected dsDNA.

In a second aspect, the present invention relates to a method for in vitro targeted sequence- specific double-stranded DNA enrichment or depletion, the method comprising: (a) contacting a sample, preferably a biological sample, containing double-stranded DNA (dsDNA) with a catalytically inactive ribonucleoprotein Cas9/crRNA tracRNA in a solution, where the catalytically inactive ribonucleoprotein Cas9/crRNA tracRNA comprises a target dsDNA sequence binding specificity, preferably a customizable target dsDNA sequence binding specificity, and optionally an affinity tag, (b) incubating the catalytically inactive

ribonucleoprotein Cas9/crRNA tracRNA with the sample under conditions sufficient for forming a complex comprising the catalytically inactive ribonucleoprotein Cas9/crRNA tracRNA and a selected dsDNA, and (c) isolating the complex from the sample. In a preferred embodiment, the ribonucleoprotein Cas9/crRNA tracRNA is rendered catalytically inactive by at least (a) at least one mutation in the Cas9 protein, where the mutation at least substantially abolishes catalytic activity, or (b) the solution is at least substantially devoid of an agent required for catalytic activity of the ribonucleoprotein Cas9/crRNA tracRNA, or (c) a catalytic inhibitor is present. Preferably, the solution lacks or substantially lacks Mg ²⁺ . In a preferred embodiment, the Cas9 protein is mutated in a RuvC active center or a HNH domain to render the nuclease catalytically inactive.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the complex is isolated from the sample using the affinity tag, the affinity tag selected from the group consisting of biotin, desthiobiotin, streptavidin, polyhistidine, maltose binding protein (MBP), chitin binding domain (CBD), and combinations thereof.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment in step (c) of the method of the invention the complex is isolated from the sample by an interaction between the affinity tag and a binding molecule, the binding molecule capable of binding with the affinity tag, and the binding molecule attached to a solid support. Preferably, the method further comprises releasing the bound complex from the solid support.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the affinity tag is attached to the Cas9 protein, the synthetic tracrRNA, the synthetic crRNA, and/or a tracrRNA crRNA hybrid duplex molecule.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the complex is isolated using an antibody having affinity for at least a portion of the catalytically inactive ribonucleoprotein Cas9/crRNA tracRNA.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment

the isolated selected dsDNA is used in targeted sequencing. Alternatively or in combination with any previous embodiments, in a further preferred embodiment following isolation and removal of the selected dsDNA from the sample, the substantially depleted of dsDNA sample is used in downstream applications.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the method of the invention results in up to 70% enrichment of the selected dsDNA from the sample.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the method of the invention results in up to 70% depletion of the selected dsDNA from the sample.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the dsDNA is a collection of fragmented dsDNA.

Alternatively or in combination with any previous embodiments, in a further preferred embodiment the catalytically inactive nuclease or catalytically inactive ribonucleoprotein retains binding affinity for the selected dsDNA.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates experimental workflow.

FIG. 2 schematically represents crRNA tracrRNA molecules used for formation of tertiary Cas9/crRNA tracrRNA complex.

FIG. 3 demonstrates catalytic activity of Cas9/crRNA tracrRNA complexes having biotinylated tracrRNA.

FIG. 4 demonstrates specific binding of biotinylated Cas9/crRNA tracrRNA to target DNA and formation of higher complexity complexes.

FIGS. 5A, 5B show gel results demonstrating capture of biotinylated

Cas9/crRNA tracrRNA using streptavidin coated magnetic beads.

FIGS. 6A, 6B show the potential of biotinylated Cas9/crRNA tracrRNA complexes in both target enrichment and target depletion.

FIG. 7 illustrates the ability of biotinylated Cas9/crRNA tracrRNA to bind specifically to the target DNA and to form higher complexity complexes in more complex DNA mixtures than shown in FIG. 4. FIG. 8 shows biotinylated Cas9/crRNA/tracrRNA complexes used for both target enrichment and target depletion as in FIG. 6, but with more complex mixture of DNA fragments.

FIG. 9 shows Cas9-RNA binding with target DNA.

FIG. 10 shows isolation of specific DNA fragments.

FIG. 1 1 is an experimental workflow of chloroplast DNA enrichment or depletion.

FIG. 12 shows relative enrichment normalized to control sample of specific DNA fragments from Arabidopsis thaliana.

DESCRIPTION

A schematic representation of crRNA tracrRNA molecules used for forming the tertiary

Cas9/crRNA tracrRNA complex is shown in FIG. 2. Cas9 protein is complexed with 42-nt-long crRNA and 74-nt-long tracrRNA which carries the biotin moiety at its very 3' end. Twenty S'- terminal nucleotides of crRNA are complementary to a 20 nucleotide length region in the target DNA, and crRNA guides Cas9 protein to its target DNA. In the vicinity of a target region a PAM sequence, required for Cas9 interaction with the double-stranded DNA, is located. PAM sequence is NGGNG for Streptococcus thermophilus (underlined) or NGG for Streptococcus pyogenes (bold) Cas9 proteins. 5'-terminal part of tracrRNA makes complementary interactions with the 3' end of crRNA, while the rest of tracrRNA potentially form secondary hairpin structures. The biotin label on tracrRNA is used for capturing of complexes with bound target DNA on streptavidin-coated magnetic beads.

FIG. 3 illustrates the catalytic activity of Cas9/crRNA tracrRNA complexes having biotinylated tracrRNA. Lane 1 - agarose gel-resolved double-stranded DNA fragments used for catalytic activity studies. Only the smallest fragment (847 bp) has a target for

Cas9/crRNA tracrRNA complex shown in Figure 2, while two others, 231 1 bp and 1310 bp in length, don't have such targets; lane 2 - reaction products resulting after the incubation of fragments shown in Lane 1 with biotin-free Streptococcus thermophilus Cas9/crRNA tracrRNA complex. The DNA fragment of 847 bp is cleaved into two fragments, 712 bp and 135 bp in length, of which the smaller one is masked by tracrRNA and crRNA which both are present in the reaction mixture; lane 3 - reaction products resulting after the incubation of fragments shown in Lane 1 with biotin-free Streptococcus pyogenes Cas9/crRNA tracrRNA; lane 4 - the same as in Lane 2 except biotin-labeled tracrRNA was used for complex formation; lane 5 - the same as in Lane 3 except biotin-labeled tracrRNA was used for complex formation. M - O'GeneRuler 1 kb Plus DNA Ladder. The results show that biotin doesn't interfere with the catalytic activity of Cas9/crRNA tracrRNA complexes.

FIG. 4 illustrates the ability of biotinylated Cas9/crRNA tracrRNA to bind specifically to the target DNA and to form higher complexity complexes. Lane 1 - agarose gel-resolved three linear DNA fragments of 231 1 bp, 1310 bp and 847 bp, where the only 847 bp fragment has a target for Cas9/crRNA tracrRNA; lane 2 - reaction products of fragments shown in Lane 1 after their incubation with Streptococcus thermophilus Cas9/crRNA tracrRNA biotinylated complex under magnesium ions-free conditions. Only change in electrophoretic mobility of the smallest fragment was observed, while the mobility of two other fragments remained unchanged; lane 3 - the same as in Lane 2 except Streptococcus pyogenes Cas9 was used for complex formation instead of Streptococcus thermophilus Cas9. M - O'GeneRuler 1 kb Plus DNA Ladder. The results show that in the case of both Cas9/crRNA/tracrRNA complexes site-specific DNA binding occurs as manifested by mobility shift of the fragment which has the target for Cas9/crRNA tracrRNA complexes used.

FIGS. 5A, 5B are results from SDS-PAGE gels illustrate the ability to capture biotinylated Cas9/crRNA tracrRNA from Streptococcus thermophilius (FIG. 5A) and

Streptococcus pyogenes FIG. 5B) using streptavidin coated magnetic beads. Lane 1 - Streptococcus thermophilus Cas9 protein as a control (160 kDa); lanes 2 and 3 -

Streptococcus thermophilus Cas9/crRNA tracrRNA biotin-free complex after incubation with streptavid in-coated magnetic beads. MB (lane 2) stands for the fraction of Cas9 bound and later on eluted from magnetic beads, while Sup (lane 3) shows Cas9 which remained in supernatant after removal of streptavidin-coated magnetic beads; lanes 4 and 5 - Streptococcus thermophilus Cas9/crRNA tracrRNA biotin-bearing complexes after their incubation with streptavidin-coated magnetic beads. MB - proteins eluted from magnetic beads, Sup - proteins found in supernatant; lane 6 - Streptococcus pyogenes Cas9 protein as a control (160 kDa); lanes 7 and 8 - Streptococcus pyogenes biotin-free Cas9/crRNA tracrRNA complexes after their incubation with streptavidin-coated magnetic beads. MB - proteins eluted from magnetic beads, Sup - proteins left in supernatant; lanes 9 and 10 - Streptococcus pyogenes biotin-carrying Cas9/crRNA tracrRNA complexes after their incubation with streptavidin coated magnetic beads. MB - proteins eluted from magnetic beads, Sup - proteins found in supernatant. M - PageRuler Plus Prestained Protein Ladder.

The results showed that the biotinylated tracrRNA acted as an affinity tag to capture Cas9 molecules which are included into tertiary complexes, while non-specific binding of biotin- free Cas9/crRNA tracrRNA complexes to streptavidin-coated magnetic beads (lanes 2 and 7) was much less efficient.

The potential of biotinylated Cas9/crRNA tracrRNA complexes in both target enrichment and target depletion is shown in FIGS. 6A, 6B from Streptococcus thermophilius (FIG. 6A) and Streptococcus pyogenes (FIG. 6B). Lane 1 - agarose gel-resolved DNA fragments of 231 1 bp, 1310 bp, and 847 bp, where the only 847 bp fragment is a substrate for Cas9/crRNA tracrRNA; lane 2 - electrophoretic analysis of DNA bound to Streptococcus thermophilus Cas9/crRNA tracrRNA biotinylated complexes which were then captured by magnetic beads and later released from magnetic beads; lane 3 - DNA fragments remaining in the supernatant after removal of magnetic beads; lanes 4 and 5 - same as in lanes 2 and 3, respectively, except that Streptococcus pyogenes Cas9 was used instead of Cas9 from Streptococcus thermophilus for complex formation. M - O'GeneRuler 1 kb Plus DNA Ladder. The results demonstrated that the fragment of interest, in this case the 847 bp in length fragment, may be either enriched (lanes 2 and 4) or depleted (lanes 3 and 5), depending on which DNA fraction, the magnetic bead-bound DNA or DNA that remained in supernatant after removal of the magnetic bead-bound DNA, was used for further studies.

FIG. 7 illustrates the ability of biotinylated Cas9/crRNA tracrRNA to bind specifically to the target DNA and to form higher complexity complexes as in FIG. 4, but in more complex DNA mixture. Lane 1 - agarose gel-resolved three linear DNA fragments of 231 1 bp, 1310 bp, and 847 bp, where the only 847 bp fragment has a target for Cas9/crRNA tracrRNA; Lane 2 - agarose-gel resolved Λ DNA, digested into ten dsDNA fragments, none of which has a target for Cas9/crRNA tracrRNA; lane 3 - reaction products of fragments shown in lane 1 and lane 2 after their incubation with Streptococcus thermophilus Cas9/crRNA tracrRNA biotinylated complex under magnesium ions-free conditions. The only change in electrophoretic mobility was of the 847 bp fragment, while the mobility of other fragments remained unchanged; lane 4 - same as in lane 3 except Streptococcus pyogenes Cas9 was used for complex formation instead of Streptococcus thermophilus Cas9. M - O'GeneRuler 1 kb Plus DNA Ladder. The results showed that site-specific DNA binding, manifested by mobility shift of the fragment which has the target for Cas9/crRNA tracrRNA, occurred in a complex environment having much more non-specific DNA fragments compared to the mixture shown in FIG. 4.

FIG. 8 demonstrates the use of biotinylated Cas9/crRNA tracrRNA complexes in both target enrichment and target depletion, as in FIG. 6, but using more complex mixture of DNA fragments. Lane 1 - agarose gel-resolved DNA fragments of 231 1 bp, 1310 bp, and 847 bp, where the only 847 bp fragment is a substrate for Cas9/crRNA tracrRNA; lane 2 - agarose-gel resolved Λ DNA, digested into ten fragments, none of which has Cas9/crRNA tracrRNA target; lane 3 - agarose-gel resolved mixture of fragments shown in Lanes 1 & 2; Lane 4 - electrophoretic analysis of DNA bound to Streptococcus thermophilus Cas9/crRNA tracrRNA biotinylated complexes which were then captured by magnetic beads and later on released from magnetic beads; lane 5 - DNA fragments left in the supernatant after removal of magnetic beads; lanes 6 and 7 - the same as in lanes 4 and 5, respectively, except that Streptococcus pyogenes Cas9 was used instead of Cas9 from Streptococcus thermophilus for complex formation. M - O'GeneRuler 1 kb Plus DNA Ladder. Results demonstrate that the fragment of interest, in this case the one of 847 bp in length, may be either enriched (lanes 4 and 6) or depleted (lanes 5 and 7) using more complex mixtures of DNA fragments.

Example I

To show that Cas9/tracrRNA crRNA complexes containing biotin moiety were catalytically active, i.e. cleaved target DNA in a sequence specific manner, plasmid pMTC- eGFP-N (4468 bp) was digested into three fragments (231 1 , 1310, and 847 bp) using Fast Digest restriction endonucleases BamHI, Mfel (Muni), and Rsrll (Cpol). Only one fragment (847 bp length) had a target for Cas9/tracrRNA crRNA. For Cas9/crRNA tracrRNA complex formation, first crRNA tracrRNA duplex (10 μΜ) was made by annealing equimolar amounts of tracrRNA and crRNA in 10 mM Tris-HCI (pH 7.5 at 37°C), 100 mM NaCI, 1 mM EDTA, then mixing with 1.55 μΜ Cas9 protein and 10X complex formation buffer (100 mM Tris-HCI (pH 7.5 at 37°C), 1000 mM NaCI, 10 mM EDTA, 0.5 mg/ml BSA, 10 mM DTT) and incubating for one hour at 37°C for the complex to form. 100 ng of plasmid DNA fragments were incubated with 250 ng Cas9/tracrRNA crRNA complexes, one made using Streptococcus thermophilus Cas9 and the other made with Streptococcus pyogenes Cas9, in a 20 μΙ reaction volume for five min at 37°C in a reaction buffer containing Mg ²⁺ (10 mM Tris-HCI (pH 7.5 at 37°C), 100 mM NaCI, 10 mM MgCI ₂ , 0.05 mg/ml BSA, 1 mM DTT), and then cleaned by adding one volume phenol- chloroform, vortexing, spinning, and taking the upper phase. Reaction products were mixed with 6X DNA loading dye and used for DNA electrophoresis in the 1 % agarose gel containing 5 μg ml ethidium bromide in 1X TAE buffer.

Results are shown in FIG. 3. Biotin did not interfere with the catalytic activity of complexes and cleavage occurred in a sequence specific manner; only the fragment having Cas9/crRNA tracrRNA target was cleaved into 712 bp and 135 bp fragments, whereas the other two fragments remained uncut.

Example II

To show that Cas9/tracrRNA crRNA forms complexes with target DNA in the absence of Mg ²⁺ ions in a sequence specific manner, the DNA of the same digested plasmid as in Example I was used. 100 ng of digested plasmid was incubated with 500 ng

Cas9/tracrRNA crRNA complex (using Cas9 from both Streptococcus thermophilus and Streptococcus pyogenes) for one hour at 37°C in the same reaction buffer as in Example I except lacking Mg ²⁺ . The reaction mixture was then mixed with 6X DNA loading dye and loaded into 1 % agarose gel with 5 μg ml ethidium bromide in 1X TAE buffer.

Result are shown in FIG. 4. There was a shift of the fragment bearing the

Cas9/tracrRNA crRNA target, presumably due to Cas9/tracrRNA crRNA binding to it and forming higher complex of lowered electrophoretic mobility. In contrast, mobility of target-free fragments remained unchanged.

Example III

The potential of using biotinylated Cas9/tracrRNA crRNA complexes for specific DNA fragment isolation using streptavidin coated magnetic beads was demonstrated by the following two experiments.

In the first experiment, 500 ng biotinylated Cas9/crRNA tracrRNA complexes and non- biotinylated complexes as controls were mixed with 5 μΙ TBST buffer (25 mM Tris-HCI, 0, 15 M NaCI, 0,05 % Tween-20, pH 7,47) prewashed streptavidin-coated magnetic beads (Pierce, Thermo Scientific), suspended in 20 μΙ PBS buffer (137 mM NaCI, 2.3 mM KCI, 4.3 mM Na ₂ HP0 ₄ , 1.76 mM KH ₂ P0 ₄ , pH 7.4). This mixture was incubated for 15 min at 4°C with constant mixing. After attraction of the magnetic beads using a magnetic rack, the supernatant was transferred to another tube, while the magnetic beads were washed with 1 ml TBST buffer and suspended in 20 μΙ PBS buffer. Both samples were mixed with 5X protein loading dye and 20X DTT and heated at 95°C for five min. The beads were separated from the reaction mixture and both the supernatant and the sample released from beads were loaded into 10% PAA gel in 1X SDS-glycine buffer. After protein electrophoresis, gels were stained with PageBlue Protein Staining Solution (Thermo Scientific). The results demonstrated that Cas9 from biotin-free Cas9/crRNA/tracrRNA complexes did not bind in a non-specific manner to streptavidin-coated magnetic particles. When biotin- bearing tracrRNA was used, Cas9 was found in the fraction eluted from the magnetic beads.

Collectively, these results clearly indicate that the Cas9/crRNA tracrRNA complex was captured by streptavidin-coated magnetic beads, and capturing depended on the presence of the biotin moiety on tracrRNA (FIG. 5).

In the second experiment, Cas9/tracrRNA crRNA complexes were incubated with fragments of the same digested plasmid in the buffer without magnesium ions as described in Example II and mixed with 5 μΙ of streptavidin-coated magnetic particles (pre-washed with 1 ml of TBST buffer and suspended in 20 μΙ PBS buffer). Reaction mixtures were incubated for 15 min at 4°C with constant mixing. The tubes were placed into a magnetic rack and the supernatant was transferred to fresh vials. Magnetic beads presumably containing bound Cas9/crRNA tracrRNA were suspended in nuclease free water and heated for five min at 70°C to disrupt streptavidin-biotin interaction and to release bound DNA fragments from

Cas9/tracRNA crRNA complexes. The magnetic particles were then placed into a magnetic rack, and the water which contained the eluted DNA was transferred to the new vial.

Supernatant and the collected elution mixture were mixed with 6X DNA loading dye and loaded into 1 % agarose gel with 5 μg ml ethidium bromide in 1 X TAE buffer.

The results shown in FIG. 6 demonstrated that only the desired fragment was isolated and the other two fragments remained in the supernatant.

Example IV

To demonstrate that Cas9/tracrRNA crRNA forms higher complexes with DNA fragment in a sequence specific manner in the absence of magnesium ions, the experiment described in Example II was repeated using additional DNA of higher complexity. 100 ng of the same digested plasmid as in Example I was mixed with 200 ng of FastDigest Styl (Eco130l) digested Λ DNA (10 fragments). The mixture of DNA fragments was incubated with 500 ng Cas9/tracrRNA crRNA complex (using Cas9 from both Streptococcus thermophilus and Streptococcus pyogenes) for one hour at 37°C in the same reaction buffer as in Example I except without Mg ²⁺ . Subsequently, the reaction mixture was mixed with 6X DNA loading dye and loaded into 1 % agarose gel with 5 μg ml ethidium bromide in 1 X TAE buffer.

Results, shown in FIG. 7, indicated shift of the fragment possessing the target for Cas9/tracrRNA crRNA, likely due to Cas9/tracrRNA crRNA binding to it, and forming a higher- complexity complex of lowered mobility. Other non-specific fragments did not exhibit a shift.

Example V

The potential of using biotinylated Cas9/tracrRNA crRNA complex for specific DNA fragment isolation from complex mixture using streptavidin-coated magnetic beads was shown by the following experiment.

100 ng of the same digested plasmid as in Example I was mixed with 200 ng

FastDigest Styl (Eco130l) digested Λ DNA (10 fragments) and incubated with

Cas9/tracrRNA crRNA complexes in buffer without Mg ²⁺ described in Example IV. Five μΙ streptavidin-coated magnetic particles (pre-washed with 1 ml of TBST buffer and suspended in 20 μΙ PBS buffer) was then added to Cas9/crRNA/tracrRNA and DNA mixture. The reaction mixtures were incubated for 15 min at 4°C with constant mixing. The tubes were placed into a magnetic rack and the supernatant was transferred to fresh vials. The magnetic beads presumably containing bound Cas9/crRNA tracrRNA were suspended in nuclease free water and heated for five min at 70°C to disrupt streptavidin-biotin interaction and to release bound DNA fragments from Cas9/tracRNA crRNA complexes. Magnetic particles were then placed into a magnetic rack, and the water which contained the eluted DNA was transferred to a new vial. Supernatant and the collected elution mixture were mixed with 6X DNA loading dye and loaded into 1 % agarose gel with 5 μg ml ethidium bromide in 1X TAE buffer.

Results, shown in FIG. 8, demonstrated that only the specific desired fragment was isolated, while all other DNA fragments remained in the supernatant.

Example VI

Homing endonuclease l-Scel is chemically labeled with biotin and incubated with the mixture of double-stranded DNA fragments, of which only one contains the target for l-Scel, under magnesium-free conditions. Formation of the enzyme:DNA complex is visualized by observing the shift in electrophoretic mobility. The enzyme together with the bound DNA fragment is captured by streptavidin-coated magnetic beads, and subsequently the bound DNA is eluted from the magnetic particles. Gel electrophoresis of eluted fragments reveals that only the fragment possessing the target for l-Scel is extracted from the mixture of DNA fragments, indicating that homing endonucleases and their mutants of altered specificity may be explored in DNA enrichment or depletion experiments.

Example VII

TALEN meganuclease of predesigned specificity is chemically labeled with biotin and is incubated with a mixture of double-stranded DNA fragments, of which only one contains the target for TALEN meganuclease, under magnesium-free conditions. Formation of the enzyme:DNA complex is visualized by observing the shift in electrophoretic mobility. The enzyme together with the bound DNA fragment is captured by streptavidin-coated magnetic beads, and subsequently the bound DNA is eluted from magnetic particles. Gel

electrophoresis of eluted fragments reveals that only the fragment possessing the target for TALEN meganuclease is extracted from the mixture of DNA fragments, indicating that TALEN meganucleases of designed specificity may be explored in DNA enrichment or depletion experiments.

Example VIII

Catalytically inactive TFO - nuclease of predesigned specificity is chemically labeled with biotin and is incubated with a mixture of double-stranded DNA fragments, of which only one contains the target for TFO - nuclease. Formation of the enzyme: target DNA complex is visualized by observing the shift in electrophoretic mobility. The enzyme together with the bound DNA fragment is captured by streptavidin-coated magnetic beads, and subsequently the bound DNA is eluted from magnetic particles. Gel electrophoresis of eluted fragments reveals that only the fragment possessing the target for TALEN meganuclease is extracted from the mixture of DNA fragments, indicating that TALEN meganucleases of designed specificity may be explored in DNA enrichment or depletion experiments.

Example IX

Cas9-RNA binding with target DNA analysis

200 ng of digested Λ DNA (10 fragments) were mixed with digested plasmid pMTC- eGFP-N (231 1 , 1310, and 847 bp; the 847 bp fragment was the Cas9-RNA target). The mixture of DNA fragments was incubated with 500 ng biotin-tagged Cas9-RNAcomplex for one hour at 37°C in the above described buffer in the absence of Mg ⁺² . Cas9-RNA complexes were prepared as described in Karvelis et al. Biochem. Soc. Trans. 41 (2013) 1401-1406. The reaction mixture was then mixed with 6X DNA loading dye and analyzed on agarose gel.

Plasmid pMTC-eGFP-N (4468 bp) was digested into three fragments (231 1 , 1310, and 847 bp) using Fast Digest restriction endonucleases BamHI, Mfel (Muni) and RsrII (Cpol). Only the 847 bp fragment contained the target for Cas9-RNA. Λ DNA was digested into ten fragments using FastDigest Styl (Eco130l). Fragments from both plasmid and Λ DNA were mixed and incubated with Cas9-RNA complexes assembled with S.thermophilus or

S. pyogenes Cas9 proteins in a buffer without Mg ⁺² . Analysis was performed in 1 % agarose gel.

FIG. 9 shows electrophoretic mobility of the 847 bp fragment was altered (arrows), while the mobility of other fragments remained unchanged. These results show that site- specific DNA binding, shown by mobility shift of the fragment that contained the target for Cas9-RNA, occurred in a complex environment having many non-specific DNA fragments. These results showed that both S. thermophilus and S. pyogenes Cas9 proteins bound specifically to target DNA under Mg-free conditions.

Example X

Isolation of specific DNA fragments

Six pi of prewashed streptavidin-coated magnetic particles were incubated with dsDNA Cas9-RNA complexes for 15 min at 4°C under constant mixing. The supernatant was removed, magnetic beads with bound dsDNA Cas9-RNA were washed with TBST, resuspended in nuclease-free water, and heated for five min at 70°C to disrupt streptavidin- biotin interaction and release bound DNA fragments. The eluate was loaded into agarose gels or sequenced using MiSeq (lllumina).

The same digested plasmid and Λ DNA were used as described in Example IX. They were mixed and incubated with Cas9-RNA complexes assembled with both Cas9 proteins in a buffer without Mg ⁺² . The Cas9 proteins with bound target DNA were captured using streptavidin-coated magnetic beads.

FIG. 10 shows that only the 847 bp fragment was captured and eluted from magnetic beads (lane MB), while other non-specific fragments remained in supernatant (lane sup). Specific fragment isolation was observed with both Cas9 proteins from S. thermophilus and S. pyogenes. Biotinylated dsDNA Cas9-RNA complexes were captured by streptavidin-coated magnetic beads.

Example XI

Chloroplast DNA enrichment from total A. thaliana DNA using S. thermophilus Cas9. A. thaliana DNA was purified using Thermo Scientificrt, GeneJETIF Plant Genomic

DNA Purification Mini Kit (Thermo Fisher Scientific). Fifty ng DNA was fragmented using MuSeek Library Preparation Kit, lllumina TM compatible. Seven crRNAs targeting chloroplast DNA with a minimum number of off-targets in genomic DNA were chosen according to Gibbs free energy calculations (Di et al. Cell. 152 (2013)1 173-83). Isolated DNA fragments were PCR amplified and size selected before sequencing on MiSeq (lllumina). Bioinformatical analysis was done using scripts in Python with some functions from HTSeq (EMBL

Heidelberg). Experimental workflow of chloroplast DNA enrichment or depletion from total A. thaliana DNA is shown in FIG. 1 1.

Total DNA from A.thallana was extracted and fragmented. Seven targets in chloroplast DNA were chosen and complementary crRNAS (Chl_1-7) were synthesized. Chl_1 and Chl_5 had two targets in DNA because of inverted repeat sequences in the chloroplast genome. Cas9-RNA complex with multiple crRNAs and S. thermophilus Cas9 was made and used to capture specific DNA fragments. dsDNA Cas9-RNA complexes were then isolated using magnetic beads. Eluate and supernatant were prepared for next generation sequencing with MiSeq (Illumine) platform.

A. thaliana DNA was purified using Thermo Scientific™ GeneJET™ Plant Genomic DNA Purification Mini Kit. Purified DNA was fragmented using MuSeek Library Preparation Kit, lllumina™ compatible (Thermo Fisher Scientific) and purified with Thermo Scientific™

MagJET™ NGS Cleanup and Size Selection Kit, protocol A. One hundred ng DNA was incubated with Cas9/tracrRNA crRNA complexes. For Cas9/crRNA tracrRNA complex formation, first crRNA tracrRNA duplexes (10 μΜ) were made by annealing equimolar amounts of tracrRNA and crRNA in 10 mM Tris-HCI (pH 7.5 at 37°C), 100 mM NaCI, 1 mM EDTA solution. Seven different crRNAs were mixed in proportions depending on target number in A.thaliana chloroplast DNA up to total 10 μΜ concentration and used for crRNA tracrRNA duplex formation. crRNA tracrRNA duplexes were mixed with 0.6 equimolar amount of

Streptococcus thermophilus Cas9 and complex formation buffer (10 mM Tris-HCI (pH 7.5 at 37°C), 100 mM NaCI, 1 mM EDTA, 0.05 mg/ml BSA, 1 mM DTT) and incubated for one hour at 37°C. One hundred ng DNA fragments were incubated with 490 ng Cas9/tracrRNA/crRNA complexes in a 20 μΙ reaction volume for one hour at 37°C in a reaction buffer containing 10 mM Tris-HCI (pH 7.5 at 37°C), 100 mM NaCI, 0.05 mg/ml BSA, 1 mM DTT). Five μΙ streptavidin-coated magnetic particles (pre-washed with 1 ml of TBST buffer and suspended in 20 μΙ PBS buffer) was then added to Cas9/crRNA/tracrRNA and DNA mixture. The reaction mixtures were incubated for 15 min at 4°C with constant mixing. The tubes were placed into a magnetic rack and the supernatant was transferred to fresh vials. The magnetic beads presumably containing bound Cas9/crRNA/tracrRNA were resuspended in 1 ml of TBST buffer, the tubes were placed into a magnetic rack, and the supernatant was removed. Magnetic beads were suspended in nuclease free water and heated for five min at 70°C to disrupt streptavidin-biotin interaction and release bound DNA fragments from Cas9/tracRNA/crRNA complexes. Magnetic particles were then placed into a magnetic rack, and the water with eluted DNA was transferred to a new vial. Eluted DNA was purified with Thermo Scientific™ MagJET™ NGS Cleanup and Size Selection Kit, protocol A. Isolated DNA fragments were PCR amplified with MuSeek Indexes and MuSeek Library Preparation Kit, lllumina™ compatible (Thermo Fisher Scientific). After amplification, DNA fragments were size selected using Thermo Scientific™ MagJET™ NGS Cleanup and Size Selection Kit, protocol C. Three hundred μΙ of Binding Mix was used for size selection procedure. The same procedure except for treatment with Cas9/crRNA tracrRNA complex was performed with control sample. DNA libraries were quantified with KAPA Library Quantification Kit (Kapa Biosystems) and sequenced with lllumina MiSeq. Relative enrichment was determined by comparing number of reads of specific sequence in sample treated with Cas9/crRNA tracrRNA complex and control sample.

FIG. 12 shows relative enrichment, normalized to control sample and calculated from bioinformatics analysis data, of specific DNA fragments from Arabidopsis thaliana total DNA fragment mixture was achieved using the inventive method and could be used in more complex applications. Chl_1 - Chl_7 represents seven different crRNAs and their targets in A. thaliana chloroplast DNA. The results demonstrated feasability of enrichment of specific DNA fragments from a chloroplast DNA fragment mixture. Chloroplast DNA from A.thaliana total DNA mixture was successfully isolated using multiple crRNAs, with enrichment varying from 0 to 42 times compared to control.

The above Examples show that Cas9-RNA complexes were successfully used for isolation of specific DNA fragments from a DNA fragment mixture as shown by digested plasmid and chloroplast DNA enrichment.

The embodiments shown and described in the specification are only specific embodiments of inventors who are skilled in the art and are not limiting in any way. Therefore, various changes, modifications, or alterations to those embodiments may be made without departing from the spirit of the invention in the scope of the following claims. The references cited are expressly incorporated by reference herein in their entirety.