Methods and kits for targeted genome enrichment

Abstract

Disclosed is a method for cleaving a target DNA to isolate a target DNA fragment sequence of interest, guided by a targeting oligonucleotide, followed by enrichment of the target DNA fragment sequence. The targeting oligonucleotide binds to the target DNA during DNA cleavage. After cleavage, the target DNA fragment sequence of interest is modified using a linking or polymerase extension method. The resulting target DNA fragment sequence of interest is enriched by exonuclease treatment.

Description

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 596,263, filed on November 4, 2023, which is entitled “METHOD AND REAGENT KIT FOR TARGETED GENOMIC ENRICHMENT” and is incorporated herein by reference in its entirety. All references cited and discussed in this specification are incorporated herein by reference in their entirety, and to the same extent as if each reference was individually incorporated by reference. Field of the Invention

[0002] The present invention relates to methods and kits for isolating and amplifying dsDNA fragments from larger dsDNA fragments or genomic dsDNA with sequence specificity. One of the applications of the present invention is for targeted genome enrichment, for example, to isolate a DNA region of interest from a whole genome for DNA sequencing. Background Art

[0003] Advances in next-generation sequencing technologies have increased our ability to sequence large genomes at lower costs and faster speeds than ever before. However, it is still not feasible to routinely apply whole genome sequencing in clinical settings. The main reason is that the cost and time of sequencing the entire genome with an accuracy level sufficient to read out the variants of interest are still prohibitively high. Contrary to the common concept that a person only needs to undergo genetic sequencing once in his or her lifetime, multiple sequencing may be required, each time for a specific purpose. For example, in cancer diagnosis, heterogeneous cell populations (such as tumor cells and normal cells) will be sequenced at the same time. When analyzing disease progression, cells from the same source may need to be sequenced at different times. Sequencing can also be applied to prenatal diagnosis of specific cell populations.

[0004] In many applications, the goal is simply to obtain an accurate image of one or certain regions of the genome of these specific cell populations. Without isolating specific genomic regions, whole genome sequencing is not only wasteful, but also leads to delays and inaccuracies. Therefore, genomic enrichment methods that allow isolation of one or more specific regions of interest will significantly reduce sequencing costs, improve accuracy, and shorten the time to result.

[0005] Many methods have been used to perform genomic enrichment. One method is based on PCR, in which multiple PCR primers are designed and tested. However, PCR amplification and standardization processes are labor-intensive, and therefore, this method cannot be generally applied. In addition, PCR can only be used for DNA fragments of certain limited size ranges, and the complexity of the genome makes it difficult to achieve high-multiplicity PCR with consistent results. The second method is based on sequence-specific ligation followed by universal PCR. Similarly, ligation probe design, process optimization and size restrictions make it less ideal. The third method is based on microarray hybridization. Genomic DNA is cut into small fragments, and a subset of genomic DNA sequences is captured based on complementary sequence identity. The captured DNA fragments are then obtained by a typical library construction scheme.

[0006] A common feature of existing targeted genomic enrichment methods is that if the DNA region of interest is longer than a few hundred bases, it is captured in the form of small fragments no longer than a few hundred bases. In PCR-based methods, the length of each fragment is limited by the ability to reliably and consistently PCR amplify the fragment, and is usually several hundred bases long. In hybridization methods, genomic DNA is randomly sheared into fragments of about a few hundred bases in length, and each fragment is then captured by hybridization. There are many inherent problems in capturing long DNA regions of interest in small fragments: (1) not all fragments are captured with the same efficiency, and some fragments may be lost completely, and (2) many probes will have to be designed and manufactured to cover the entire length of the region of interest, resulting in higher costs. In addition, PCR may introduce errors into the amplified fragments. For hybridization, specificity is low and the processing time is long. Summary of the invention

[0007] The key to overcoming the shortcomings of existing targeted genomic enrichment methods is the ability to specifically cleave and isolate long DNA regions of interest in large fragments, preferably as one whole fragment, rather than separating many short fragments as in current methods. This requires the ability to (1) cleave the target DNA at a predetermined site with sequence specificity and (2) isolate the cleaved DNA region of interest.

[0008] Described herein are methods and kits for lysing and purifying 5x10 2 -1x10 8The invention relates to a method for preparing a DNA fragment of interest with eight base pairs, so that targeted genome enrichment and selective genome sequencing can be performed with higher specificity, simpler workflow and lower cost. The core of the invention is a sequence-specific DNA nuclease that can cleave target DNA with sequence specificity, protection of the targeted DNA segment after nuclease cleavage, removal of non-target DNA and separation of cleaved DNA fragments using exonucleases. Sequence specificity means that engineered nucleases can cleave DNA with eight base pairs or better sequence specificity. A specific sequence must be present for engineered nuclease cleavage. The cleavage point may or may not be precisely located at any specific base, but will be near its position guided by the targeting sequence. Non-specific background cleavage may also be present.

[0009] In one embodiment of the invention, a method uses a sequence-specific DNA nuclease. The nuclease includes one or more targeting oligonucleotides. The nuclease is capable of specifically cleaving the target double-stranded DNA with a length greater than eight base pairs. The cleaved DNA has a sticky end. The cleaved DNA segment of interest has a sticky end flanking the target DNA. In another embodiment, a method for cutting out a DNA fragment of interest from a target DNA comprises: cleaving the target DNA with the sequence-specific DNA nuclease described above; adding modified nucleotides to the sticky ends by a polymerase, or adding a linker with a modified base to the end by a ligase; removing unmodified DNA with an exonuclease; and labeling the enriched target DNA for further purification and manipulation.

[0010] In another embodiment, the kit includes a sequence-specific DNA nuclease, wherein the DNA nuclease is capable of specifically cleaving the target double-stranded DNA with a sequence length greater than eight base pairs; a polymerase, wherein modified nucleotides are added to the sticky ends; or a ligase, wherein linkers with modified bases are added to the target DNA; an exonuclease, wherein non-target DNA is removed from the reaction mixture; and an enzyme, wherein a tag is added to the enriched target DNA.

[0011] describe BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a diagram of the disclosed method for enriching one or more large genomic dsDNA fragments of a prokaryotic or eukaryotic organism.

[0013] Figure 2 shows a schematic diagram of sequence-specific nuclease gRNA / Cas12a protein binding to a hypothetical target DNA and forming double-stranded breaks flanking a single target region.

[0014] Figure 3The results of an experiment performed as shown in Figure 2 are illustrated, demonstrating the protection of the two plasmid dsDNA fragments generated after gRNA / Cas12a cleavage.

[0015] Figure 4 A diagram of the structure of the human ABL proto-oncogene 1 (ABL1) is illustrated.

[0016] FIG. 5 presents the results after enrichment of ABL1 DNA fragments isolated from human genomic dsDNA samples using GAPDH as a control. DETAILED DESCRIPTION

[0017] General Definition

[0018] The term "Cas12a-associated guide RNA" refers to an RNA oligonucleotide that binds to the Cas12a protein and recognizes a target DNA region of interest and guides the Cas12a nuclease there for editing.

[0019] The term "Cas12a protein" refers to Cas12a (CRISPR-associated protein 12a, formerly known as Cpf1), and it is a subtype of the Cas12 protein and forms part of the CRISPR system in some bacteria and archaea.

[0020] The terms "sticky" and "sticky ends" refer to double-stranded DNA (dsDNA) having unpaired (single-stranded) DNA nucleotides on the 5'-strand or 3'-strand and are referred to as short overhangs. Figure 2C In the figure, 105 and 105' are respectively illustrated, which are modified and natural nucleotides, respectively, and the modified and natural nucleotides are filled in the short overhang to provide complementary chains.

[0021] The terms "cut," "cleavage," and "cleaved" refer to the creation of breaks in a dsDNA strand through the sugar-phosphate backbone of the DNA strand.

[0022] The term "enrichment" or "enriched" refers to an increase in the concentration of DNA fragments of interest compared to non-DNA of interest within a DNA sample.

[0023] The term "exonuclease" refers to an enzyme capable of digesting single-stranded and / or double-stranded DNA, including but not limited to exonuclease III, T7 exonuclease, exonuclease V, exonuclease VIII, lambda exonuclease, T5 exonuclease, nuclease Bal-31, variants and truncated forms thereof, and combinations thereof.

[0024] As used herein, the term "genomic DNA" refers to double-stranded DNA (dsDNA) from a cell, tissue or culture sample of prokaryotic or eukaryotic origin.

[0025] The term "guide RNA" (gRNA) refers to an RNA fragment that acts as a guide for an RNA-targeting enzyme or a DNA-targeting enzyme, such as, but not limited to, a CRISPR endonuclease, with which the guide RNA forms a complex. The complex is capable of cleaving DNA at specific sites on, within, at, or flanking a target DNA sequence within a genomic DNA sample.

[0026] The term one or more "ligases" refers to one or more enzymes capable of ligating oligonucleotides and / or nucleotide sequences to the 5' or 3' end of a DNA and / or RNA molecule or sequence. Ligase may include, but is not limited to, T4 DNA ligase, T4 DNA ligase 2, T4 RNA ligase 1, T4 RNA ligase 2, SplintR ligase, RtcB ligase, T3 DNA ligase, TaqDNA ligase, 9°N DNA ligase, E. coli DNA ligase, and variants and truncated forms thereof and combinations thereof.

[0027] The term "ligation" refers to the covalent joining of two ends of a DNA or RNA molecule.

[0028] The term "linker" refers to a double-stranded DNA or RNA molecule that can be covalently attached to the end of the double-stranded DNA or RNA molecule.

[0029] The term "modified" refers to oligonucleotides, nucleotides, etc. that are chemically modified in the triphosphate portion, sugar portion, or base thereof. These nucleotides include, but are not limited to, α-phosphorothioate nucleoside triphosphates, morpholino triphosphates, peptide nucleic acids, peptide nucleic acid analogs, and sugar-modified nucleoside triphosphates and combinations thereof.

[0030] The term "predetermined" means defined or determined in advance.

[0031] The terms "protect" and "protected" mean kept safe or protected from undesirable enzymatic treatment, including but not limited to digestion and / or another chemical, physical or mechanical treatment or exposure means.

[0032] The terms "specific" and "specificity" refer to a property of being uniquely belonging to or relating to a target DNA sequence or DNA sequence fragment.

[0033] The term "sequence specific" refers to a well-defined region of a DNA or RNA strand / sequence.

[0034] In one embodiment, a method for enriching target DNA is disclosed. In a single sample having at least one target DNA fragment sequence and at least one specific DNA nuclease having a targeting oligonucleotide (ON), the targeting oligonucleotide (ON) is homologous to their respective selected binding sites on the target double-stranded DNA ("dsDNA") (e.g., FIG. 2A to FIG. 2D As used herein, homology means that the targeting ON is complementary to one strand on the target dsDNA, and is therefore capable of forming a triple helix with the target dsDNA, or a double helix with a complementary single DNA strand. The sequence-specific DNA nuclease binds to the target DNA at the binding site, forming a target DNA-DNA nuclease complex, and the sequence-specific DNA nuclease cuts the target DNA at the cleavage point, which is on or near the binding site of the targeting oligonucleotide of the target dsDNA. After cutting the target DNA at the 5' end and the 3' end (when it is dsDNA, these two ends of the complementary strands, or if it is single-stranded DNA, only the 5' or 3' strand), a polymerase is used to incorporate natural nucleotide bases ( Figure 2D , 105') and modified nucleotide bases ( Figure 2D , 105), including but not limited to α-phosphorothioate nucleoside triphosphates, morpholino triphosphates, peptide nucleic acids, peptide nucleic acid analogs, and sugar-modified nucleoside triphosphates. Figure 2D , 106) one or more modified nucleotide bases are attached to both ends of the fragment of interest, the target fragment is dsDNA, and if it is ssDNA, it is attached to the single strand. These modified bases can prevent DNA from being digested by DNA exonucleases. Non-target linear DNA in the sample is not protected by modified nucleotide bases at both ends, and therefore, the unprotected non-target DNA is digested by exonucleases. This allows the target DNA fragment of interest to be enriched in the final reaction mixture (e.g. Figure 1 Detailed and Figure 3 and Figure 5 ).

[0035] In one aspect of this embodiment, as shown in FIG2 , the reaction includes targeting oligonucleotides 104 and 104′, which are guide RNAs or their analogs. Nucleases 102 and 102′ include CRISPR-associated protein 12a (Cas12a) or one or more variants thereof (Advancedseq LLC, Livermore, CA USA). The targeting oligonucleotide guides the Cas12a protein or its variants to introduce double-strand breaks in the target DNA 110. The use of Cas12a protein 102 or 102′ as a programmable sequence-specific DNA endonuclease is described in Ledford H. “Bacteria yield new gene cutter”. Nature. 526 (7571): 17. doi: 10.1038 / nature.2015.18432. PMID 26432219, (October 2015), which is incorporated herein by reference.

[0036] As used herein, Cas12a variants include Cas12a mutants that maintain some or all of the Cas12a functions, or Cas12a homologs derived from a common ancestor that perform the same or similar functions as Cas12a. Figure 2B As described, by incubating with CRISPR / Cas12a or a variant thereof, the target DNA 112 is cleaved at both ends. The targeting oligonucleotide is present in a suitable buffer (10-100 mM Tris, pH 6-8) at a suitable temperature (25-42° C.) for a suitable length of time (5 minutes-4 hours). In a preferred embodiment, the target DNA 112 is released from other non-target DNA 108, 108′ (e.g. Figure 2B as described).

[0037] In another aspect of the embodiment, multiple pairs of sequence-specific DNA nucleases are used in one reaction. Therefore, multiple different sequence-specific DNA fragments covering multiple sequence-specific regions of a DNA sequence of interest can be cut and separated in multiple reactions in a single vial.

[0038] In another aspect of the embodiment, multiple pairs of sequence-specific DNA nucleases are used in one reaction to cleave the same DNA sequence of interest from the same target DNA but at different cleavage points, thereby generating multiple fragments of interest, all of which include the same DNA region of interest. By performing such redundant cleavages on the same DNA sequence of interest, the overall efficiency, i.e., the percentage of target DNA cleavage, can be increased. By combining the two aforementioned methods, such as Figure 1 2 , multiple DNA fragments covering the same DNA sequence of interest as well as multiple DNA sequences of interest can be cut and separated in one run.

[0039] Since Cas12a endonuclease produces sticky ends after cleavage, the polymerase 106 ( Figure 2D ) The modified nucleotides are added to the sticky ends of the cracked DNA segment of interest for enrichment as the next step. Many polymerases are capable of incorporating modified nucleotide bases that are complementary to the template strand of the original DNA. These polymerases include, but are not limited to, Phusion DNA polymerase, Taq DNA polymerase, Vent DNA polymerase, Bst DNA polymerase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, Therminator DNA polymerase, DNA polymerase I, Klenow Fragment, T4 DNA polymerase, T7 DNA polymerase, Bsu DNA polymerase, and terminal transferase.

[0040] Modified nucleotides may include, but are not limited to, triphosphates, including α-phosphorothioate nucleoside triphosphates, morpholino triphosphates, peptide nucleic acids, peptide nucleic acid analogs, and / or sugar-modified nucleoside triphosphates.

[0041] In the enrichment step, the unmodified DNA is digested with an exonuclease including, but not limited to, Exonuclease III, T7 Exonuclease, Exonuclease V, Exonuclease VIII, λ Exonuclease, T5 Exonuclease, Nuclease Bal-31, and variants and truncated forms.

[0042] All publications, published patent documents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated to be incorporated by reference.

[0043] Example

[0044] Embodiment 1

[0045] Enrichment of plasmid pGEM3Zf DNA using the claimed method

[0046] This approach has been experimentally demonstrated for the first time in plasmid pGEM-3Zf. Figure 3 In the experiment described, the pGEM-3Zf plasmid was digested with CRISPR / Cas12a in the presence of two guide RNAs to release two DNA fragments of 0.7 and 2.5 kb, respectively (lane 1). The guide RNA is complementary to the end of the target genomic DNA fragment that is longer than eight base pairs, so the probability of finding a matching complementary genomic DNA sequence is 262144 (4 9 =262144) bases. The 5'-short overhang of the cleavage fragment was filled by incorporating dNTPs into the sticky ends at 72°C for 30 minutes (data not shown). The modified fragments were then subjected to exonuclease III digestion. To confirm that the exonuclease was able to digest the unprotected DNA, purified bacteriophage lambda DNA (0.5 μg) was added to the reaction mixture and then subjected to exonuclease treatment (lane 2).

[0047] Figure 3 , Lane 3 shows that the exonuclease digestion did not digest the protected ends. The modified fragments were then subjected to exonuclease digestion with phage lambda DNA (lane 3). This resulted in complete digestion of the phage lambda DNA when the exonuclease was added, but the pGEM-3Zf plasmid 0.7 kb and 2.5 kb fragments remained undigested. This result demonstrates that the disclosed and claimed sequence-specific DNA enrichment method is effective for plasmid DNA.

[0048] Example II

[0049] Isolation of ABL1 as a target gene from human genomic dsDNA using the claimed method

[0050] To confirm that the CRISPR / Cas12a-based enrichment strategy can be used to selectively enrich long DNA sequences from the actual human genome, we selected human ABL1 as the target gene for the validation experiment. The ABL1 gene is approximately 175-kb long, such as Figure 4 The ABL1 gene is composed of multiple intronic and exonic fragments. As a proto-oncogene encoding a protein tyrosine kinase, the ABL1 gene is involved in various cellular processes, including cell division, adhesion, differentiation, and response to stress. Notably, ABL1 has also been found to be fused to several translocation partner genes, most notably the breakpoint cluster region gene (BCR). BCR-ABL fusion proteins have been found in many forms of leukemia, including in most cases of chronic myeloid leukemia (CML). Therefore, the ABL1 gene has become an ideal disease biomarker and therapeutic target for CML and other related leukemias.

[0051] In order to cleave the ABL1 gene from human genomic DNA, 1 μg of total DNA was isolated from cultured HEK293 cells treated with CRISPR / Cas12a and ABL-5'-guide RNA and ABL-3'-guide RNA complexes at 37°C for 30 minutes. After cleaving the genomic DNA with CRISPR-based Cas12a / guide RNA complexes, a mixture of dNTP analogs and 10 units of Taq DNA polymerase were added to the cleavage reaction mixture at a final concentration of 25 μM each. After incubation at 72°C for 30 minutes, the reaction product was purified using a ZYMO DNA purification kit (DNA Clean Kit) (ZYMO Research, Tustin, CA, USA) according to the manufacturer's instructions. The purified DNA product was eluted with TE buffer to a final concentration of 20 ng / μl.

[0052] After filling the 5'-short overhang of the DNA fragment cleaved by CRISPR / Cas12a with α-phosphorothioate deoxyribonucleotides and purifying with ZYMO DNA purification kit, the isolated DNA product was treated with exonuclease III (New England Biolabs) according to the manufacturer's instructions. Each reaction was carried out in a total volume of 20 μl, containing about 0.1 μg of isolated DNA and 50 units of exonuclease III. The reaction mixture was incubated at 37 ° C for 30-60 minutes, followed by incubation at 70 ° C for 15 minutes to remove the remaining exonuclease activity. The final product was then analyzed by real-time quantitative PCR assay of genomic DNA samples.

[0053] In the TaqMan real-time qPCR assay, each qPCR reaction consisted of an initial incubation at 94°C for 5 minutes, followed by 40 amplification cycles, each cycle at 94°C for 10 seconds and at 60°C for 40 seconds. The qPCR had sequence-specific primers / probes for ABL1 (probe labeled with Cy5) and housekeeping gene GAPDH (probe labeled with FAM), respectively, so the assay was able to detect both genes simultaneously within a single reaction. Figure 5A As shown, the signals of ABL1 (purple - middle trace) and GAPDH gene (blue - upper trace) were detected simultaneously in the same TaqMan qPCR reaction before exonuclease III treatment. The Ct value of ABL1 was 25, and the Ct of GAPDH was 25.6. As expected, after treatment of DNA with exonuclease III, only the signal of ABL1 gene was not affected in the multiplex assay (Ct = 25), while the unprotected GAPDH gene lost most of its signal (Ct = 34) ( Figure 5B ).like Figure 5A and Figure 5B Ct values ​​were determined from increasing points in the respective traces for ABL1 and GAPDH, as indicated by the vertical lines through the traces.

[0054] The relative change in Ct values ​​between the TaqMan assays was determined using the Ct values ​​from 5A and 5B (ΔCt = 0.4 for ABL1 and ΔCt = 9 for GAPDH with and without exonuclease treatment). Since the difference in Ct values ​​for GAPDH was greater than 8, it was estimated that ABL1 was enriched at least 100-fold relative to GAPDH in this experiment (2 8 = 256-fold difference, since each unit difference in Ct value represents a 2-fold difference in concentration). These results demonstrate that the claimed method can effectively separate and enrich large genomic dsDNA segments from human genomic DNA.

[0055] In another embodiment, the target DNA can be modified in the enrichment step by adding an adapter or tag, including but not limited to, for example, biotin or another affinity tag for binding the target DNA to a solid support and pulling down the target DNA from the reaction solution mixture. The resulting isolated target DNA can undergo further purification and manipulation to perform, for example, sequencing analysis as known to the skilled artisan.

[0056] Although embodiments and applications of the present disclosure have been shown and described, the terms and the following claims should not be construed as limiting the claims to the specific embodiments of the disclosed specification. It will be apparent to those skilled in the art that many more modifications and improvements than those mentioned above are possible without departing from the inventive concept herein. All possible embodiments and equivalents of the full range should be understood with respect to the disclosed terms and such claims to which rights are granted, so that the claims are not limited by the present disclosure.

Claims

1. A method for improving the specificity of enriching target DNA, the method comprising: a. providing a genomic target DNA sample, the genomic target DNA sample comprising one or more double-stranded (DS) target fragments within at least one target region, wherein the at least one target region comprises one or more double-stranded target DNA fragments; b. cleaving the two ends of the one or more double-stranded target fragments with a CRISPR / Cas12a-guide RNA (gRNA) endonuclease complex, wherein the endonuclease is a sequence-specific DNA nuclease; and wherein the gRNA is complementary to each flanking region inside or outside the one or more target fragments to be enriched at its corresponding end, thereby forming a sticky 5' short overhanging single-stranded end; c. incorporating modified nucleotides (ON) into each of the 5' short overhangs of the one or more DS target fragments using a DNA polymerase, wherein the modified nucleotides prevent exonuclease digestion of the one or more DS target fragments; and d. Digesting the unprotected genomic DNA with an exonuclease, wherein the one or more target DS fragments are specifically enriched.

2. The method of claim 1, wherein each of the Cas12a-gRNA complexes comprises a Cas12a protein and a Cas12a-related gRNA, and the Cas12a protein and the Cas12a-related gRNA are complementary to different predetermined sites of the targeted genomic DNA.

3. The method of claim 1, wherein the sequence-specific DNA nuclease is capable of cleaving a target double-stranded DNA (dsDNA) with sequence specificity as determined by the guide RNA, the guide RNA being complementary to the ends of the target genomic DNA fragments longer than eight base pairs, such that the probability of finding a matching complementary genomic DNA sequence is 262144 (4 9 = one of 262144) bases.

4. The method of claim 1, wherein the sequence-specific DNA nuclease coupled with its associated guide RNA is capable of generating one or more sticky ends that cleave double-stranded target DNA fragments.

5. The method of claim 1, wherein the DNA polymerase is capable of modifying the sticky ends of the cleaved target DNA fragments by attaching the modified ON to the sticky ends, thereby protecting one or more of the target DS fragments thereof from DNA exonuclease digestion.

6. The method of claim 1, wherein the exonuclease is capable of enriching one or more target DNA fragments, wherein the one or more exonucleases digest non-target DNA regions and fragments that are not modified ON-protected at both ends.

7. The method of claim 1, wherein the enriched one or more target DNA fragments can be further modified to facilitate purification for sequence analysis.

8. The method of claim 1, wherein the modified nucleotide is selected from the group consisting of α-phosphorothioate nucleoside triphosphates, morpholino triphosphates, peptide nucleic acids, peptide nucleic acid analogs, and sugar-modified nucleoside triphosphates, and combinations thereof.

9. The method of claim 7, wherein the enriched one or more target DNA fragments are further purified for sequence analysis.

10. The method of claim 1, wherein the sequence-specific DNA nuclease comprises a Cas12a protein or a variant thereof.

11. The method of claim 1, wherein the guide RNA is single-stranded DNA or RNA.

12. The method of claim 1, wherein each of the gRNAs is between 15-100 nucleotides in length, and wherein a 10-50 nucleotide long sequence is complementary to a sequence on one strand at each end of the target DNA fragment.

13. The method of claim 1, further comprising at least one pair of targeting oligonucleotides, and the composition allows the target DNA to be cleaved by the sequence-specific DNA nuclease at both ends of the DNA fragment of interest in a manner such that both ends are sticky.

14. The method of claim 4, wherein the sticky ends generated by cleavage by sequence-specific DNA nuclease have a short 5' overhang.

15. The method of claim 1, further comprising at least one polymerase and modified nucleotides, and the polymerase is capable of incorporating modified nucleotides at the sticky ends of the target DNA.

16. The method of claim 1, further comprising at least one ligase and a double-stranded DNA or RNA adapter or a hairpin adapter having modified nucleotides, and the ligase and the adapter are capable of ligating to the ends of the enriched target DNA.

17. A kit comprising a. Sequence-specific DNA nuclease; b.DNA polymerase; c. modified nucleotides; d. DNA exonuclease; and optionally e. Ligase.

18. The kit of claim 17, wherein the sequence-specific DNA nuclease is capable of cleaving target double-stranded DNA with sequence specificity greater than eight base pairs in length and generating sticky ends.

19. The kit of claim 17, wherein the DNA polymerase is capable of filling in the sticky ends with modified nucleotides after cleavage of the target dsDNA fragment by the DNA nuclease.

20. The kit of claim 17, wherein the DNA exonuclease is capable of digesting DNA without the protection of modified nucleotides at both ends.

21. The kit according to claim 17, wherein the DNA ligase is capable of ligating a DNA adapter, an RNA adapter, or an adapter having a modified nucleotide base to both ends of one or more of the enriched target DNAs.

22. A DNA enrichment kit, comprising at least two of the following reagents: a.Cas12a protein or its variants; b. Targeting oligonucleotide (guide-RNA); c.DNA polymerase; d. modified nucleotides; e. DNA exonuclease; f. DNA or RNA ligase; and g. DNA / RNA linker.

23. A method for isolating a target dsDNA using a kit as described in any one of claims 17 and 22, the kit comprising a targeting oligonucleotide and a sequence-specific DNA nuclease, the targeting oligonucleotide being a single-stranded DNA or RNA and being 15-100 nucleotides in length, the nuclease specifically cleaving the target DNA, and wherein a sequence of 10-50 nucleotides in length is substantially complementary to a sequence on one strand of the target dsDNA.

24. The kit of any one of claims 17 and 22, wherein the DNA polymerase is selected from the group consisting of: Phusion DNA polymerase, Taq DNA polymerase, Vent DNA polymerase, Bst DNA polymerase, phi29 DNA polymerase, Sulfolobus DNA polymerase IV, Therminator DNA polymerase, DNA polymerase I, Klenow fragment, T4 DNA polymerase, T7 DNA polymerase, Bsu DNA polymerase and terminal transferase.

25. The kit of any one of claims 17 and 22, wherein the modified nucleotide is selected from the group consisting of α-phosphorothioate nucleoside triphosphates, morpholino triphosphates, peptide nucleic acids, peptide nucleic acid analogs or sugar-modified nucleoside triphosphates and combinations thereof.

26. The kit of any one of claims 17 and 22, wherein the exonuclease is selected from the group consisting of Exonuclease III, T7 Exonuclease, Exonuclease V, Exonuclease VIII, λ Exonuclease, T5 Exonuclease, Nuclease Bal-31, variants thereof, and truncated forms thereof.

27. The kit of any one of claims 17 and 22, wherein the ligase is selected from the group consisting of T4 DNA ligase, T4 DNA ligase 2, T4 RNA ligase 1, T4 RNA ligase 2, SplintR ligase, RtcB ligase, T3DNA ligase, Taq DNA ligase, 9°N DNA ligase, Escherichia coli DNA ligase, variants thereof, and truncated forms thereof.

28. The kit of any one of claims 17 and 22, wherein the DNA linker may comprise any combination of DNA nucleotide bases, RNA nucleotide bases and modified nucleotide bases; and wherein, They may be in double-stranded linear form or have a hairpin structure.

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