Fusion protein comprising nucleic acid binding protein and method for capturing specific nucleic acid using same

Abstract

The present invention relates to a fusion protein comprising a nucleic acid binding protein and a method for capturing a specific nucleic acid using same. The fusion protein is formed by fusing a cytosine base editor and a uracil binding protein. The method is a method for capturing, from a sample, DNA (containing a PAM and containing cytosine C at a specific position) containing a specific target sequence, and comprises: contacting a sample containing target DNA with the fusion protein to capture the target DNA, and detecting the target DNA. Under the mediation of a sgRNA library, provided is a use of the fusion protein in capturing, from a sample, DNA containing a specific target sequence and / or detecting the DNA. Target DNA can be captured from a complex nucleic acid sample with high throughput and high sensitivity and specificity, and practical application value is provided.

Description

Nucleic acid binding protein fusion protein and method for capturing specific nucleic acid Technical Field

[0001] This article relates to a method for capturing a target nucleic acid, and more particularly to a fusion protein of a nucleic acid-binding protein and a method for capturing a specific nucleic acid. Background Art

[0002] There are currently a variety of methods and applications for capturing target nucleic acids from polynucleotide samples (e.g., in a whole genome). Due to the influence of complex DNA samples (e.g., affecting the signal-to-noise ratio), it is not conducive to the specific recognition of the target sequence, and the target sequence needs to be captured. In research work, gene capture probes are used to capture a specific region. A probe sequence of a certain length is designed for this region. Each sequence moves a certain distance along the gene position, and then a large amount of the above sequence is synthesized artificially to sequence the captured DNA product (Calvo SE, Compton AG, Hershman SG, et al. Molecular diagnosis of infantile mitochondrial disease with targeted next-generation sequencing. Sci Transl Med, 2012, 4(118):118-110). The current methods for capturing specific nucleic acids involve multiple steps and require the use of a large amount of nucleic acid samples. At the same time, the capture process is cumbersome, difficult, time-consuming and labor-intensive, with low accuracy and high cost, and requires expensive instruments and equipment. For example, targeted DNA hybridization capture technology requires the design of specific single-stranded DNA with a length of more than 100 bp, and a complex hybridization process with the sample nucleic acid under specific conditions, which can take up to more than ten hours or even longer. However, prolonged hybridization incubation significantly affects the capture process. In addition, higher incubation temperatures and longer incubation times will affect the salt ion concentration in the mixture, especially when the hybridization reaction volume is small. For example, the target DNA capture rate of existing solid-phase or liquid-phase targeted DNA hybridization capture technologies can only reach ~400 times (Enrichment of sequencing targets from the human genome by solution hybridization. Ryan Tewhey, Masakazu Nakano, Xiaoyun Wang, Carlos Pabón- Barbara Novak, Angelica Giuffre, Eric Lin, Scott Happe, Doug N Roberts, Emily M LeProust, Eric J Topol, Olivier Harismendy and Kelly A Frazer. Genome Biol. 2009; 10(10): R116. doi: 10.1186 / gb-2009-10-10-r116) and ~1000-fold (Microarray-based genomic selection for high-throughput resequencing. David T Okou, Karyn Meltz Steinberg, Christina Middle, David J Cutler, Thomas J Albert & Michael E Zwick. Nat Methods. 2007 Nov; 4(11): 907-9). Moreover, this process is costly, has low specificity, and has low DNA capture efficiency. Moreover, the capture effect is greatly affected by the experimental skills of the personnel and cannot be automated. Therefore, it is necessary to develop a new method for capturing specific nucleic acids that is fast, efficient, and low-cost.

[0003] In addition, single nucleotide variations (SNVs) account for approximately 58% of human pathogenic genetic variations (Rabinowitz R, Abadi S, Almog S, et al. Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing. Nucleic Acids Res, 2020, 48(W1): W340-W347). With the increasing understanding of the impact of genomic DNA sequences on human health, the application potential of genome editing technology in clinical treatment has become apparent. Single base editors (BEs) based on the CRISPR-Cas system have shown broad clinical application prospects. This editor can efficiently and accurately repair SNVs at specified sites, but BEs may cause off-target editing at other sites in the genome that are similar to the targeted site. As a therapeutic approach, genome editing tools must have high targeted editing efficiency and minimal harmful or non-targeted editing. Existing methods for assessing the specificity of CRISPR-Cas systems in eukaryotic cells rely on either computational prediction of potential off-target sites based on sequence similarity between targeted and off-target sites or require whole-genome sequencing. Bioinformatics-based computational methods can only predict potential off-target sites, while whole-genome sequencing is expensive and difficult to achieve high-throughput detection of off-target effects in BEs. Therefore, a new method for high-throughput, low-cost detection of off-target editing is needed.

[0004] Summary of the Invention

[0005] The present invention first provides a fusion protein, which is formed by the fusion of a cytosine base editor and a uracil-binding protein, wherein the cytosine base editor is UdgX, and the uracil-binding protein is a BE3 editor, or its optimized SSBE3 editor.

[0006] The uracil-binding protein is UdgX protein from M. smegmatis, M. avium, R. imtechensis, M. haemophilum, Rhodococcus spp., Streptomyces coelicolor, Gordonian amibiense, Bradyrhizobium japonicum and Nocardia farcidia. More specifically, its amino acid sequence is shown in SEQ ID No. 22.

[0007] The cytosine base editor is a base editor that can produce uracil U after base editing;

[0008] Preferably, the cytosine base editor is a cytosine base editor that is a BE3-type editor, or a preferred BE3-type editor, specifically preferably a post-BE3-type editor that is SSBE3, whose amino acid sequence is shown in SEQ ID NO: 23.

[0009] The uracil binding protein and the cytosine base editor are directly connected or connected through a linker. Preferably, the amino acid sequence of the linker is shown in SEQ ID NO: 24. In a specific embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID No. 1.

[0010] In some embodiments, the fusion protein comprises a sequence having motif A (GEQPG) and motif B (HPSSLL) in UdgX and contains the conserved region KRRIH. In some embodiments, the fusion protein can be a protein comprising or consisting of the amino acid sequence encoded by SEQ ID No. 1 or SEQ ID No. 2, or a variant thereof.

[0011] In some embodiments, the fusion protein can be a protein comprising or consisting of an amino acid sequence that is approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID No. 1 or the amino acid sequence encoded by SEQ ID No. 2. In some embodiments, a variant of the fusion protein has one or more unnatural amino acids, one or more amino acid substitutions, one or more amino acid insertions, one or more amino acid deletions, or any combination thereof at one or more sites compared to the fusion protein. In some embodiments, the variant has substantially similar or comparable activity to the fusion protein. In some embodiments, a variant of the UdgX-SSBE3 protein can have approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identity to the amino acid sequence of the fusion protein. As known to those skilled in the art, variants of proteins can be obtained by introducing conservative substitutions, deletions and additions of amino acids when preparing recombinant proteins. Required substitutions, deletions or insertions can also be provided by changing specific codons of the coding sequence. In addition, protein variants can also be prepared by random or saturation mutagenesis techniques such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis and oligonucleotide-guided mutagenesis. In some embodiments, conservative substitutions include substitutions between amino acids of similar properties, such as substitutions between hydrophobic amino acids Nle, Met, Ala, Val, Leu, Ile, substitutions between neutral hydrophilic amino acids Cys, Ser, Thr, Asn, Gln, substitutions between acidic amino acids Asp, Glu, substitutions between basic amino acids His, Lys, Arg, substitutions between amino acids that affect chain orientation Gly, Pro, and substitutions between aromatic amino acids Trp, Tyr, Phe. In some embodiments, non-conservative substitutions between the above types can be included.

[0012] In some embodiments, the fusion protein further comprises a purification tag, thereby facilitating easy separation and / or purification. In some embodiments, the purification tags that can be used include Snap-tag, His-tag, Flag-tag, MBP-tag, biotin, and other tags commonly used for protein purification.

[0013] The present invention also provides a polynucleotide encoding the fusion protein, an expression vector and a recombinant host cell. Specifically, the polynucleotide sequence is shown in SEQ ID No. 2.

[0014] Furthermore, the present invention provides a method for capturing specific nucleic acids, and more specifically, relates to a method and application thereof for using the fusion protein to simultaneously generate uracil and a protein that binds to uracil to capture a specific target sequence DNA (containing PAM and cytosine C at a specific position). The term "containing PAM and cytosine C at a specific position" refers to a PAM sequence of NGG or NG or other types, with a length of 8-30 bp, preferably a 20 bp NGG sequence before the PAM. The term "containing cytosine C at a specific position" refers to the 3rd to 25th base upstream of the 5' end of the PAM site, preferably the 5th to 20th base, and more preferably the 11th to 17th base.

[0015] In one aspect, provided herein is a method for capturing target DNA from a sample, the method comprising:

[0016] 1) contacting a polynucleotide sample containing the target DNA with a specific fusion protein having base editing and uracil binding functions and an sgRNA targeting the target sequence to obtain a fusion protein-target DNA complex ( FIG1 );

[0017] 2) Detect and capture target DNA.

[0018] In a specific embodiment, in step 1) of the method, a target sequence DNA to be captured (containing PAM and cytosine C at a specific position) is selected, and an sgRNA targeting the target DNA is used to position the fusion protein to the target sequence so that the target base C in the target codon is edited to U. The fusion protein can be covalently bound to the target base U in the target sequence, thereby obtaining a fusion protein-target DNA complex.

[0019] In a specific embodiment, step 1) of the method further comprises washing the fusion protein-target DNA complex at least three times, preferably washing with PBS buffer or Tris-HCl (pH 8.0) at least three times.

[0020] In a specific embodiment, step 2) of the method further comprises binding the fusion protein-target DNA complex to an affinity matrix, preferably magnetic beads, more preferably Snap magnetic beads.

[0021] In some embodiments, a polynucleotide sample containing a target DNA can be mixed with a fusion protein and a corresponding sgRNA, and the mixture can be brought into contact to capture the target DNA. In some embodiments, a solution containing a fusion protein and a corresponding sgRNA can be added to a polynucleotide sample containing a target DNA, or the sample can be added to a solution containing a fusion protein and a corresponding sgRNA, and the mixture can be brought into contact to capture the target DNA.

[0022] In some embodiments, the captured sample may contain multiple different nucleic acid samples containing target editing sites. A mixed library containing sgRNAs targeting multiple target DNA strands is added to a capture reaction system containing the UdgX-SSBE3 fusion protein and target DNA, enabling high-throughput editing and simultaneous capture of multiple target DNA strands in a single reaction.

[0023] In some embodiments, the captured sample may contain long target nucleic acid samples (greater than 1KB). By designing multiple sgRNAs (at least 1 sgRNA / KB), a mixed library containing sgRNAs targeting multiple target DNAs can be added to a capture reaction system containing UdgX-SSBE3 fusion protein and target DNA, enabling high-throughput editing and capture of multiple target DNAs in a single reaction. The captured samples can be spliced ​​into complete long target DNA fragments (greater than 1KB) through second-generation sequencing.

[0024] In some embodiments, the captured sample may contain multiple target DNAs and long target nucleic acid samples (greater than 1KB) at the same time. By designing multiple sgRNAs, a mixed library of sgRNAs targeting all target DNAs can be put into a capture reaction system containing UdgX-SSBE3 fusion protein and target DNA, so that multiple target DNAs can be edited and captured at high throughput in one reaction. After the captured samples are spliced ​​together by second-generation sequencing, all target DNA sequence information can be obtained, including the spliced ​​long target DNA (greater than 1KB).

[0025] In a preferred embodiment, in step 1) of the method, a sample containing the target sequence DNA is contacted with the fusion protein and a combination of different types of targeting sgRNAs to obtain a uracil-binding protein-target DNA complex; the sgRNA combination is a mixed sgRNA library targeting multiple sites;

[0026] Specifically, the concentrations of each sgRNA were 0.1ng / ul-1ng / uL, 0.2ng / ul-2ng / uL, and 2ng / ul-20ng / uL;

[0027] Preferably, the sgRNA combination used is designed in such a way that at least one sgRNA is designed for every 1KB of target sequence captured; the more sgRNAs are used to capture every 1KB of target sequence, the higher the capture amount in the batch of captured target fragments;

[0028] In the simultaneous capture of multiple targets, the distribution density of sgRNA is increased; optionally, for target sites with low second-generation sequencing coverage, multiple corresponding sgRNAs are designed and deployed to increase the capture of target fragments;

[0029] Further preferably, the library of the sgRNA combination is obtained by using batch synthesis of sgRNA or batch forward mixed primers and specific reverse primers to amplify the corresponding sgDNA combination library, and then detecting it in a high-throughput manner by batch transcription to obtain the library of the sgRNA combination;

[0030] Also preferably, in step 1) of the method, for simultaneously capturing multiple dispersed target fragments or capturing a long target fragment that needs to be divided into multiple smaller fragments and then spliced, the library of sgRNA combinations in the capture reaction system is mixed and put into batches.

[0031] In some embodiments, the target DNA is a double-stranded DNA or single-stranded DNA containing a target sequence DNA (containing PAM and cytosine C at a specific position) or a mixture of the two. In some embodiments, the target DNA contains one or more naturally occurring or artificially added target sequence DNA (containing PAM and cytosine C at a specific position). In some embodiments, the target DNA is exogenous DNA or endogenous DNA relative to the subject. In some embodiments, the target DNA is from humans, microorganisms, animals or plants. The method of the present invention can advantageously achieve the capture and detection of target DNA present in a sample at a lower abundance. The direct purpose of the method of the present invention is to capture target DNA in a sample, that is, to solve the problem of obtaining low-abundance target DNA in a sample.

[0032] In some embodiments, the fusion protein can include a protein subunit that specifically binds uracil, such as the UdgX protein. In some embodiments, the UdgX-SSBE3 protein specifically binds to the uracil obtained by base editing without binding to other bases or with a greater affinity for uracil than for binding to other bases. According to the prior art, one of ordinary skill in the art can determine that the uracil-binding protein has a strong affinity for uracil in DNA. (A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily, Pau Biak Sang, Thiruneelakantan Srinath, Aravind Goud Patil, Eui-Jeon Woo and Umesh Varshney. Nucleic Acids Research. 30; 43(17): 8452-63. doi: 10.1093 / nar / gkv854.).

[0033] In some embodiments, the method includes separating a complex comprising the fusion protein and the target DNA to obtain the target DNA. In some embodiments, the complex comprising the fusion protein and the target DNA can be separated by any suitable separation method. For example, in some embodiments, the complex comprising the fusion protein and the target DNA can be separated from a complex nucleic acid sample using affinity separation techniques, and the captured target DNA can then be recovered. The specific binding interaction between molecules is called affinity, and the technique of purifying biomolecules using affinity is called affinity separation. In some embodiments, separation is performed based on the difference in affinity between the stationary phase-based ligand and the target molecule and its affinity for other molecules. In some embodiments, the complex comprising the fusion protein and the target DNA can be obtained by chromatography, such as affinity column chromatography. In some embodiments, the complex comprising the uracil-binding protein and the target DNA can be obtained by magnetic beads, such as affinity magnetic beads. In some embodiments, the fusion protein has a capture tag to facilitate separation and / or purification. In some embodiments, capture tags that can be used include Snap-tag, His-tag, Flag-tag, MBP-tag, biotin, and other tags commonly used for protein capture. In some embodiments, a complex of the fusion protein and the target DNA can be obtained by affinity separation using Snap magnetic beads (e.g., Snap magnetic beads commercially available from NEB). In some embodiments, after the complex of the fusion protein and the target DNA is bound to the magnetic beads or affinity column, it is washed at least three times with a lysis buffer. In some embodiments, the lysis buffer is PBS buffer or Tris-HCl (pH 8.0). It has been found that after removing impurities, it is further preferred to perform cross-linking, thereby facilitating the capture and / or detection of the target DNA with improved sensitivity and specificity.

[0034] In some embodiments, the method may further include the step of decrosslinking the fusion protein-DNA fragment complex. In some embodiments, the step can be accomplished by, for example, adding a protein-degrading protease, such as proteinase K, to the eluate of the resulting fusion protein-DNA fragment complex. In some embodiments, the method may further include amplifying the captured target DNA. Methods for amplifying target nucleic acids are widely known in the art and include, for example, PCR amplification, isothermal amplification, and the like.

[0035] In some embodiments, the source of the sample is not particularly limited, as long as it is a sample that may contain the target DNA. In some embodiments, the sample includes a genomic DNA sample, a cell-free DNA sample, an environmental genome sample and / or a mixed genomic DNA sample. In some embodiments, the sample includes double-stranded DNA, single-stranded DNA or a mixture of the two. In some embodiments, the sample can be a polynucleotide sample such as a sample containing mixed nucleic acids. In some embodiments, the sample can come from various organisms, such as humans, plants, animals, microorganisms, etc. In some embodiments, the sample can come from bacteria, archaea, protozoa, fungi, etc. In some embodiments, the sample can be an environmental genome sample (metagenomic sample). In some embodiments, the sample can come directly or indirectly from a subject. For example, the sample can be collected directly from a subject, or can come from an isolated sample obtained from a subject. In some embodiments, the sample can come from an animal subject such as a mammalian subject. In some embodiments, the sample can come from primates, experimental animals, farm animals, livestock or pets. In some embodiments, the sample can come from mammals, marine animals, amphibians, birds, reptiles, insects and other invertebrates. In some embodiments, the sample can be from a human subject. In some embodiments, the sample can be from the mucous membrane secretions, vaginal secretions, milk, tears, ascites of the subject's blood, serum, serosal fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, secretory tissues and organs, for example, from the liquid of the pleura, pericardium, peritoneum, abdomen or other body cavities. In some embodiments, the sample can be to obtain cell-free DNA from subject's body fluids such as plasma or serum. In some embodiments, the sample can be a genomic DNA sample separated from the above-mentioned sources. In some embodiments, the sample can be a mixed sample obtained from multiple sources, for example, a mixed genomic DNA sample. In some embodiments, the sample can be a bacterial and / or human genomic DNA sample. In some embodiments, the target DNA is in the fetal cell fraction of cell-free DNA, and wherein the cell-free DNA is from maternal plasma.

[0036] In some embodiments, capturing target DNA refers to the process of obtaining a higher percentage of target DNA in a polynucleotide population. In some embodiments, the percentage of target DNA increases by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more. In some embodiments, the percentage of target DNA increases by about 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold. In some embodiments, the methods of the present invention can be used to capture uracil-containing target DNA more than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000 or more times. In some embodiments, the methods of the present invention can be used to capture and / or detect target DNA from samples with low target DNA content. For example, target DNA is captured and / or detected from a sample in which the ratio of target DNA to total DNA in the sample is 1 / 400, 1 / 500, 1 / 600, 1 / 700, 1 / 800, 1 / 900, 1 / 1000, 1 / 2000, 1 / 3000, 1 / 4000, 1 / 5000, 1 / 6000, 1 / 7000, 1 / 8000, 1 / 9000, 1 / 10000, 1 / 20000, 1 / 30000, 1 / 40000, 1 / 50000, 1 / 60000, 1 / 70000, 1 / 80000, 1 / 90000, 1 / 100000, 1 / 200000, 1 / 300000, 1 / 400000, 1 / 500000 or less. In some embodiments, the methods of the present invention can capture and / or detect target DNA containing as little as 0.2% (or less) of a genetic mutation in a sample containing about 99.8% (or more) of non-mutant DNA. In some embodiments, it has been found that the methods of the present invention can capture and / or detect target DNA from complex nucleic acid samples with high sensitivity and / or specificity.

[0037] In some embodiments, provided herein is a method for detecting a target DNA, comprising obtaining captured target DNA by the methods described herein, and then detecting the target DNA. In some embodiments, the method comprises amplifying the captured target DNA after obtaining the captured target DNA. In some embodiments, the presence and / or sequence of the target DNA can be detected by methods such as hybridization, PCR, isothermal amplification, sequencing, and / or biochips.

[0038] In some embodiments, the fusion protein is used to capture and / or detect target DNA from a sample. In some embodiments, the fusion protein or a composition containing a base editor-pyrimidine binding protein is used to capture and / or detect target sequence DNA (containing PAM and cytosine C at a specific position) from a sample. In some embodiments, the fusion protein is used to prepare a composition and / or kit for capturing and / or detecting target sequence DNA (containing PAM and cytosine C at a specific position). In some embodiments, it relates to the use of a fusion protein in the preparation of a reagent for capturing target sequence DNA (containing PAM and cytosine C at a specific position) from a sample. Preferably, the fusion protein is as defined above, the target DNA is as defined above, and the sample is as defined above.

[0039] In some embodiments, the high-throughput capture method for specific nucleic acids provided herein can be used to detect off-target sites of different base editors in different samples. By using the high-throughput capture method for specific nucleic acids, samples captured by fusion proteins containing different types of base editors in different samples are obtained, and DNA sequence information of non-target sites is obtained by second-generation sequencing, thereby detecting the off-target sites of the base editor in the sample.

[0040] The method of the present invention can capture target DNA from complex nucleic acid samples with high sensitivity and specificity, and can simultaneously capture multiple target DNAs and long target DNA fragments. Further through optimization, the method of the present invention can capture target DNA from complex nucleic acid samples with high throughput, high sensitivity and high specificity, and can simultaneously capture multiple target DNAs and long target DNA fragments. It can also be used for off-target site detection and has great application value. Among them, the existing technologies on the market for targeted capture and enrichment technology are mainly based on hybridization technology, and the technology that only uses a fusion protein for capture has not been reported so far. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A: UdgX-SSBE3 protein captures DNA containing target sequence.

[0042] Figure 1B: High-throughput capture of DNA containing target sequences by UdgX-SSBE3 protein.

[0043] Figure 2: Expression and purification of UdgX-SSBE3 protein.

[0044] Figure 3: DNA sample concentration standard curve.

[0045] Figure 4: q-PCR quantitative analysis of DNA.

[0046] Figure 5: q-PCR quantification of captured target DNA in the E. coli genome.

[0047] Figure 6: q-PCR quantification of captured target DNA in the Streptomyces genome.

[0048] Figure 7: Effect of different sgRNA input amounts on the relative DNA capture rate.

[0049] Figure 8: q-PCR quantitative analysis of multiple target DNAs captured simultaneously in the Streptomyces genome.

[0050] Figure 9: Sequencing coverage analysis of multiple target DNAs captured simultaneously in the Streptomyces genome.

[0051] Figure 10: Effect of different sgRNA concentrations on the relative DNA capture rate.

[0052] Figure 11: q-PCR quantitative analysis of captured long fragment target DNA in the Streptomyces genome.

[0053] Figure 12: Sequencing coverage analysis of captured long fragment target DNA in the Streptomyces genome.

[0054] Figure 13: q-PCR quantification of captured target DNA in the human saliva genome.

[0055] Figure 14: Sequencing coverage analysis of simultaneously captured target DNA in the human saliva genome.

[0056] Figure 15: Effects of different sgRNA combinatorial libraries on the relative capture amount of target DNA.

[0057] Figure 16: Different sgRNA combinations capture the splicing of long target DNA sequences.

[0058] Figure 17: Relative capture of 51 newly added target DNA sites.

[0059] Figure 18: A combination of 142 sgRNAs captures all exon sequences of three genes.

[0060] Figure 19: 142 sgRNA combinations captured exon region sequencing coverage.

[0061] Figure 20: Relative capture of 34 newly added target DNA sites.

[0062] Figure 21: A combination of 176 sgRNAs captures all exon sequences of three genes.

[0063] Figure 22: Sequencing coverage of exon regions of 176 sgRNA combinations.

[0064] Figure 23: Venn diagram of non-target sequences in triplicate capture samples from 176 sgRNA combinations.

[0065] Figure 24: Sequencing coverage of non-target sequences in triplicate capture samples with 176 sgRNA combinations. DETAILED DESCRIPTION

[0066] The present invention is further described below with reference to specific examples for a better understanding of the present invention, but these examples do not constitute a limitation of the present invention.

[0067] Example 1 Editing and Capturing of Target DNA

[0068] 1.1 Expression and purification of UdgX-SSBE3 protein

[0069] The UdgX fusion protein is a uracil-binding protein with an amino acid sequence of SEQ ID NO: 22, and SSBE3 is a cytosine-based base editor optimized from the universal BE3-based base editor (amino acid sequence shown in SEQ ID NO: 23). The amino acid sequence of the two fusion proteins after being connected by a linker (SEQ ID NO: 24) is shown in SEQ ID No. 1, and the encoding nucleotide sequence is shown in SEQ ID No. 2.

[0070] The UdgX-SSBE3 gene sequence (SEQ ID No. 2) was constructed into the expression vector pSnap-taq(T7)2-His (containing a Snap tag) to obtain the plasmid pSnap-UdgX-SSBE3. The plasmid pSnap-UdgX-SSBE3 was transformed into Escherichia coli BL21. The BL21 cells containing the plasmid pSnap-UdgX-SSBE3 were cultured in LB medium containing 0.05% FeCl3 at 37°C until the OD600 reached 0.6. IPTG was added to a final concentration of 0.5 mM in the culture system and cultured overnight at 16°C. A 20 mL sample was centrifuged at 5000 rpm / min for 30 minutes, the supernatant discarded, and the pellet resuspended in 2 mL of lysis buffer (50 mM Tris-HCl buffer, 50 mM NaCl, pH 8). Lysozyme was added to a final concentration of 0.1 mg / mL, and the cells were placed on ice for 30 minutes before ultrasonic disruption. Centrifuge at 5000 rpm for 30 minutes. Take the supernatant and bind it to Snap magnetic beads (e.g., NEB) at 4°C overnight. Add 2 mL of buffer (50 mM Tris-HCl buffer, 50 mM NaCl, pH 8) to the centrifuge tube containing the magnetic beads. Gently invert the centrifuge tube several times to resuspend the magnetic beads. Magnetic separation is performed. Wash the magnetic beads three times to remove proteins not bound to the beads.

[0071] SDS-PAGE analysis of the UdgX-SSBE3 protein (Figure 2) was performed on the supernatant of the SSBE3 protein after sonication and after binding to Snap magnetic beads overnight at 4°C. Figure 2 shows that the target protein SSBE3 was present in the supernatant of the sonication supernatant, while the target protein content was also reduced in the supernatant of the protein after binding to Snap magnetic beads overnight at 4°C. This indicates that the target protein SSBE3 can bind to the Snap magnetic beads.

[0072] 1.2 Acquisition of DNA fragments

[0073] A forward primer (SEQ ID No. 3) and a reverse primer (SEQ ID No. 4) were synthesized and PCR amplified from the plasmid template to obtain a GFP fragment (SEQ ID No. 5) containing the target editing site.

[0074] A forward primer (SEQ ID No. 6) and a reverse primer (SEQ ID No. 4) were synthesized and PCR amplified from the plasmid template to obtain a GFP fragment (SEQ ID No. 7) that did not contain the target editing site.

[0075] 1.3 Acquisition of sgRNA Fragments

[0076] The forward primer (SEQ ID No. 8) and the reverse primer (SEQ ID No. 9) were synthesized and PCR amplified from the plasmid template to obtain the sgDNA fragment (SEQ ID No. 10) corresponding to the target editing site.

[0077] The sgRNA in vitro transcription kit (Yinmao Shengye) was used to transcribe the corresponding sgRNA using the sgDNA obtained in the previous step as a template.

[0078] 1.4 Editing and binding of UdgX-SSBE3 protein to DNA fragments

[0079] The UdgX-SSBE3 protein and sgRNA bound to the snap magnetic beads were added to 0.5 mL of reaction buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 1 mM Na2EDTA, 1 mM DTT, 25 ug / ml BSA), respectively, and reacted at 25°C for 5 minutes. Then, 500 ng of double-stranded DNA was added and reacted at 37°C for 2 hours.

[0080] 1.5 Obtaining target DNA fragments

[0081] After reacting at 37°C for 2 hours, gently invert the centrifuge tube several times to resuspend the magnetic beads and perform magnetic separation. Remove the reaction solution and add 1 mL of buffer (50 mM Tris-HCl buffer, 50 mM NaCl, pH 8) to the centrifuge tube containing the magnetic beads. Gently invert the centrifuge tube several times to resuspend the magnetic beads and perform magnetic separation. Repeat three times. Finally, add 500 μL of buffer (50 mM Tris-HCl buffer, 50 mM NaCl, pH 8) and add proteinase K to the eluate of the UdgX protein-DNA fragment complex to a final concentration of 100 μg / mL. Treat at 50°C for 30 minutes to decrosslink the UdgX-SSBE3 protein-DNA fragment complex and obtain the target DNA fragment.

[0082] 1.6 Quantitative analysis of DNA fragments

[0083] The cross-linked DNA fragments were quantified by q-PCR.

[0084] q-PCR reaction system: 6.4ul distilled water, 10ul SYBR.Green Realtime PCR Master Mix, 0.8ul upstream primer (SEQ ID No.11, 10uM), 0.8ul downstream primer (SEQ ID No.12, 10uM), 2ul sample solution.

[0085] q-PCR cycling conditions: 95°C for 30 s; PCR cycles (×40 cycles): 95°C for 5 s, 55°C for 10 s, 72°C for 15 s (data collection); melting curve analysis.

[0086] The DNA samples quantified by Nanodrop were diluted and the standard curve was plotted (Figure 3).

[0087] The concentrations of the standards are: 0.000001ng / ul, 0.00001ng / ul, 0.0001ng / ul, 0.001ng / ul and 0.01ng / ul.

[0088] Quantification of the target DNA after cross-linking was performed by q-PCR. The results showed that UdgX-SSBE3 could specifically capture the GFP fragment containing the target editing site, with the recovery concentration being approximately 314 times that of the GFP fragment without the target editing site (Figure 4).

[0089] Example 2 Isolation of target DNA from Escherichia coli genomic DNA samples

[0090] 2.1 Expression and purification of UdgX protein (same as Example 1)

[0091] 2.2 Acquisition of genomic DNA samples

[0092] A forward primer (SEQ ID No. 3) and a reverse primer (SEQ ID No. 4) were synthesized and PCR amplified from the plasmid template to obtain a GFP fragment (SEQ ID No. 5) containing the target editing site.

[0093] A forward primer (SEQ ID No. 6) and a reverse primer (SEQ ID No. 4) were synthesized and PCR amplified from the plasmid template to obtain a GFP fragment (SEQ ID No. 7) that did not contain the target editing site.

[0094] The genomic DNA of Escherichia coli DH5α was extracted using a genome extraction kit (Biomega).

[0095] 2.3 Acquisition of sgRNA fragments (same as Example 1)

[0096] 2.4 Editing and binding of UdgX-SSBE3 protein to DNA fragments

[0097] The UdgX-SSBE3 protein and sgRNA bound to the snap magnetic beads were added to 0.5 mL of reaction buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 1 mM Na2EDTA, 1 mM DTT, 25 ug / ml BSA) and reacted at 25°C for 5 minutes. Then, 6 ug of the double-stranded, disrupted E. coli genomic DNA and GFP fragments containing editing sites at different contents (1 ng and 10 ng) or 6 ug of the disrupted E. coli genomic DNA and GFP fragments without editing sites (500 ng) were added to 1 mL of reaction buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 1 mM Na2EDTA, 1 mM DTT, 25 ug / ml BSA) and reacted at 37°C for 2 hours.

[0098] 2.5 Obtaining the target DNA fragment (same as in Example 1)

[0099] 2.6 Quantitative analysis of target DNA fragments

[0100] The target DNA after de-crosslinking was quantified by q-PCR, and GFP was amplified from the de-crosslinked DNA using primers (SEQ ID No. 11) and primers (SEQ ID No. 12). The results showed that UdgX-SSBE3 could specifically capture GFP fragments containing the target editing site, with the total amount recovered being approximately 21.3 times (6ug genomic DNA: 10ng GFP fragments containing editing sites) and 2.1 times (6ug genomic DNA: 1ng GFP fragments containing editing sites) compared to GFP fragments without the target editing site (Figure 5). Since the ratios of GFP fragments containing editing sites and GFP fragments without editing sites in the starting mixed DNA sample were 10:500 and 1:500, respectively, the actual target DNA specific capture rates in this experiment were 1065-fold and 1050-fold.

[0101] Example 3 Isolation of target DNA from a Streptomyces genomic DNA sample.

[0102] 3.1 Expression and purification of UdgX protein (same as Example 1)

[0103] 3.2 Acquisition of genomic DNA samples

[0104] The Streptomyces genomic DNA was extracted using a genome extraction kit (Biomega).

[0105] 3.3 Acquisition of sgRNA fragments

[0106] Synthetic forward primers (SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15) and reverse primers (SEQ ID No. 9) were used to amplify the sgDNA fragments (Act, RedD, and RedN) corresponding to the target editing sites from the plasmid template by PCR.

[0107] The sgRNA in vitro transcription kit (Yinmao Shengye) was used to transcribe the corresponding sgRNA using the sgDNA obtained in the previous step as a template.

[0108] 3.4 Editing and binding of UdgX protein to DNA fragments

[0109] The UdgX-SSBE3 protein and sgRNA (Act, RedD and RedN) bound to the snap magnetic beads were added to 0.5 mL of reaction buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 1 mM Na2EDTA, 1 mM DTT, 25 ug / ml BSA), respectively, and reacted at 25°C for 5 minutes. Then, 8 ug of Streptomyces genomic DNA after double-stranding was added and reacted at 37°C for 2 hours.

[0110] 3.5 Obtaining the target DNA fragment (same as in Example 1)

[0111] 3.6 Quantitative analysis of target DNA fragments

[0112] q-PCR was used to quantify the target DNA after cross-linking was reversed. Primers (SEQ ID No. 16 and SEQ ID No. 17), primers (SEQ ID No. 18 and SEQ ID No. 19), and primers (SEQ ID No. 20 and SEQ ID No. 21) were used to amplify Act, RedD, and RedN from the reversed cross-linked DNA, respectively. The results showed that UdgX-SSBE3 could specifically capture DNA fragments containing the target editing site in the Streptomyces genome, with capture amounts of 0.007 ng (Act), 0.004 ng (RedD), and 0.01 ng (RedN) (Figure 6).

[0113] The experiment was repeated according to the method of Example 3, except that only three sgRNA concentrations (1-10 ng / uL) were added to a single reaction system (1 uL of each sgRNA) to simultaneously capture the three target DNAs. The results of this repeated experiment showed that compared to the results of Example 2, the capture efficiency of the Act gene decreased by 25%, the capture efficiency of the RadD gene decreased by 28%, and the capture efficiency of the RadN gene decreased by 23% (Figure 7).

[0114] Example 4: Simultaneous isolation of multiple target DNAs from a Streptomyces genomic DNA sample.

[0115] 4.1 Expression and purification of UdgX protein (same as Example 1)

[0116] 4.2 Acquisition of genomic DNA samples

[0117] The Streptomyces genomic DNA was extracted using a genome extraction kit (Biomega).

[0118] 4.3 Acquisition of sgRNA Fragments

[0119] Synthesize forward primers and reverse primers to obtain sgDNA fragments (Target1-Target7) corresponding to the target editing sites from the plasmid template through PCR amplification.

[0120] The sgRNA in vitro transcription kit (Yinmao Shengye) was used to transcribe the corresponding sgRNA using the sgDNA obtained in the previous step as a template.

[0121] 4.4 Editing and binding of UdgX protein to DNA fragments

[0122] The UdgX-SSBE3 protein bound to the snap magnetic beads and 10 sgRNAs (Target1-7, Act, RadD and RadN) were added to 0.5 mL of reaction buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 1 mM Na2EDTA, 1 mM DTT, 25 ug / ml BSA), and reacted at 25°C for 5 minutes. Then, 8 ug of Streptomyces genomic DNA after double-stranding was added and reacted at 37°C for 2 hours.

[0123] 4.5 Obtaining the target DNA fragment (same as in Example 1)

[0124] 4.6 Quantitative analysis of target DNA fragments

[0125] The target DNA was quantified by q-PCR after cross-linking was resolved, and primers were used to amplify the target DNA from the cross-linked DNA. The results showed that UdgX-SSBE3 could specifically capture DNA fragments containing the target editing site in the Streptomyces genome, with capture amounts of 0.019 ng (Target 1), 0.039 ng (Target 2), 0.052 ng (Target 3), 0.005 ng (Target 4), 0.03 ng (Target 5), 0.012 ng (Target 6), 0.004 ng (Target 7), 0.016 ng (Red D), 0.002 ng (Red N), and 0.006 ng (Act) (Figure 8).

[0126] 4.7 Next-generation sequencing of target DNA fragments

[0127] Using the cross-linked target DNA as a template, a second-generation library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 from Novogene. The constructed DNA library was then sent to Novogene Technology Co., Ltd. for next-generation sequencing. The sequencing results showed that all nine target sites were sequenced, with coverage ranging from 22,022 to 592,551 (Figure 9).

[0128] The experiment was repeated according to the method of Example 4, in which only 10 sgRNAs were mixed in equal volumes, a total of 1uL was added, and the concentration was diluted to 1 / 10 or 1 / 20 of the initial concentration. The sgRNA concentration in the reaction system was 1 / 10 or 1 / 20 of the initial value, and 10 target DNAs were captured simultaneously. The results of the repeated experiments showed (Figure 10): Compared with the results of Example 4, after the sgRNA was diluted 10 times, the capture efficiency of the target DNA decreased by (25.2%-98.6%). After the sgRNA was diluted 20 times, the capture efficiency of the target DNA decreased by (0.02%-1.05%). This test sgRNA input minimum amount required to infer the maximum amount of sgRNA that can be added in a single reaction. The more sgRNA that can be added, the more types and lengths of target DNA can be captured.

[0129] Example 5: Capturing long target DNA fragments (10 KB) from Streptomyces genomic DNA samples.

[0130] 5.1 Expression and purification of UdgX protein (same as Example 1)

[0131] 5.2 Acquisition of genomic DNA samples

[0132] The Streptomyces genomic DNA was extracted using a genome extraction kit (Biomega).

[0133] 5.3 Acquisition of sgRNA Fragments

[0134] Synthesize forward primers and reverse primers to obtain sgDNA fragments corresponding to the target editing sites (Target2, Target8-Target26, 20 of them) from the plasmid template through PCR amplification.

[0135] The sgRNA in vitro transcription kit (Yinmao Shengye) was used to transcribe the corresponding sgRNA using the sgDNA obtained in the previous step as a template.

[0136] 5.4 Editing and Binding of UdgX Protein to DNA Fragments

[0137] The UdgX-SSBE3 protein and sgRNA bound to the snap magnetic beads were added to 0.5 mL of reaction buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 1 mM Na2EDTA, 1 mM DTT, 25 ug / ml BSA), respectively, and reacted at 25°C for 5 minutes. Then, 8 ug of Streptomyces genomic DNA after double-stranding was added and reacted at 37°C for 2 hours.

[0138] 5.5 Obtaining the target DNA fragment (same as Example 1)

[0139] 5.6 Quantitative analysis of target DNA fragments

[0140] The target DNA after de-crosslinking was quantified by q-PCR, and the primers were used to amplify the target DNA from the de-crosslinked DNA. The results showed that UdgX-SSBE3 could specifically capture the DNA fragments containing the target editing sites in the Streptomyces genome, and the capture amount was 0.095ng (Target8), 0.111ng (Target9), 0.004ng (Target10), 0.005ng (Target11), 0.069ng (Target12), 0.035ng (Target13), 0.007ng (Target14), 0.034ng (Target15), 0.19ng (Target 16), 0.145ng(Target2), 0.005ng(Target17), 0.082ng(Target18), 0.264ng(Target19), 0.005ng(Target20), 0.016ng(T target21), 0.103ng (Target22), 0.164ng (Targe23), 0.292ng (Target24), 0.549ng (Target25) and 0.493ng (Target26) (Figure 11).

[0141] 5.7 Next-generation sequencing of target DNA fragments

[0142] Using the cross-linked target DNA as a template, a second-generation library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 from Novogene. The constructed DNA library was then sent to Novogene Technology Co., Ltd. for next-generation sequencing. The sequencing results showed that all 20 target sites were sequenced, with coverage ranging from 21,105 to 1,744,840, and could be assembled into 10KB long fragments (Figure 12).

[0143] Example 6 Simultaneous capture of long-fragment target DNA (10KB) from human saliva genomic DNA samples.

[0144] 6.1 Expression and purification of UdgX protein (same as Example 1)

[0145] 6.2 Acquisition of genomic DNA samples

[0146] Human saliva genomic DNA was extracted using a Saliva Genomic DNA Rapid Extraction Kit (Biomed).

[0147] 6.3 Obtaining sgRNA Fragments

[0148] The forward primer and reverse primer were synthesized and PCR amplified from the plasmid template to obtain the sgDNA fragment corresponding to the target editing site (Target27-Target41).

[0149] The sgRNA in vitro transcription kit (Yinmao Shengye) was used to transcribe the corresponding sgRNA using the sgDNA obtained in the previous step as a template.

[0150] 6.4 Editing and Binding of UdgX Protein to DNA Fragments

[0151] The UdgX-SSBE3 protein and sgRNA bound to the snap magnetic beads were added to 0.5 mL of reaction buffer (50 mM Tris-HCl, pH 8, 50 mM NaCl, 1 mM Na2EDTA, 1 mM DTT, 25 ug / ml BSA), respectively, and reacted at 25°C for 5 minutes. Then, 8 ug of human salivary genomic DNA after double-stranding was added and reacted at 37°C for 2 hours.

[0152] 6.5 Obtaining the target DNA fragment (same as in Example 1)

[0153] 6.6 Quantitative Analysis of Target DNA Fragments

[0154] The target DNA after cross-linking was quantified by q-PCR, and the target DNA was amplified from the cross-linked DNA using primers. The results showed that UdgX-SSBE3 could specifically capture DNA fragments containing the target editing sites in the human saliva genome, with capture amounts of 0.00005 ng (Target 27), 0.00005 ng (Target 28), 0.00008 ng (Target 29), 0.0002 ng (Target 30), 0.0006 ng (Target 31), 0.0002 ng (Target 32), 0.002 ng (Target 33), 0.0013 ng (Target 34), 0.001 ng (Target 35), 0.0003 ng (Target 36), 0.0023 ng (Target 37), 0.0012 ng (Target 38), 0.0041 ng (Target 39), 0.0037 ng (Target 40), and 0.003 ng (Target 41) (Figure 13).

[0155] 6.7 Next-generation sequencing of target DNA fragments

[0156] Using the cross-linked target DNA as a template, a second-generation library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 from Novogene. The constructed DNA library was then sent to Novogene Technology Co., Ltd. for next-generation sequencing. The sequencing results showed that all 20 target sites were sequenced, with coverage ranging from 1,000 to 2,909,520, and could be assembled into 10KB long fragments (Figure 14).

[0157] Example 7: The sgRNA mixed library was used to capture long-fragment target DNA (41KB) from human saliva genomic DNA samples.

[0158] 7.1 Expression and purification of UdgX-SSBE3 protein (same as Example 1)

[0159] 7.2 Acquisition of genomic DNA samples

[0160] Human saliva genomic DNA was extracted using a Saliva Genomic DNA Rapid Extraction Kit (Biomed).

[0161] 7.3 Obtaining sgRNA Fragments

[0162] The complete exon sequences of three childhood tumor-related genes (TRIM28, FBXW7, and NYNRIN) in human saliva genomic DNA samples (the three genes contain a total of 47 exons, one of which is approximately 41KB in length).

[0163] The sgRNA combinations required for the capture experiment are batch-transcribed from their corresponding sgDNA fragments. The corresponding forward and reverse primers are synthesized. The forward primers used are mixed with the reverse primers. The sgDNA fragment corresponding to the target editing site is amplified from the plasmid template via PCR.

[0164] The target fragment totaled 41kb, and a total of 91 sgRNAs were designed for capture, with an average interval of 450bp between each two sgRNAs.

[0165] A combination of three sgRNAs was used to capture the target fragments.

[0166] The first combination is to design 91 sgRNAs to mediate the capture of 41KB long exon fragments (on average, one sgRNA is set every 450bp, and the design method is designed according to the conventional method. For the sake of convenience, sgRNA1-sgRNA91 are used as codes).

[0167] The second combination consists of 46 sgRNAs. The number of sgRNAs in the first combination was reduced from 91 to 46 (only one of the two adjacent sgRNAs was retained). The sgRNAs used are as follows (the odd numbers are retained): sgRNA1, sgRNA3, sgRNA5, sgRNA7, sgRNA9, sgRNA11, sgRNA13, sgRNA15, sgRNA17, sgRNA19, sgRNA21, sgRNA23, sgRNA25, sgRNA27, sgRNA29, sgRNA31, sgRNA33, sgRNA34, sgRNA35, sgRNA36, sgRNA37, sgRNA38, sgRNA39, sgRNA40, sgRNA41, sgRNA42, sgRNA43, sgRNA44, sgRNA45, sgRNA46, sgRNA47, sgRNA48, sgRNA49, sgRNA50, sgRNA51, sgRNA52, sgRNA53, sgRNA54, sgRNA55, sgRNA56, sgRNA57, sgRNA58, sgRNA59, sgRNA60, sgRNA61, sgRNA62, sgRNA63, sgRNA64, sgRNA65 NA35,sgRNA37,sgRNA39,sgRNA41,sgRNA43,sgRNA45,sgRNA47,sgRNA49,sgRNA51,sgRNA53,sgRNA55,sgRNA57,sgRNA59,sgRNA61,sgRNA 63, sgRNA65, sgRNA67, sgRNA69, sgRNA71, sgRNA73, sgRNA75, sgRNA77, sgRNA79, sgRNA81, sgRNA83, sgRNA85, sgRNA87, sgRNA89 and sgRNA91.

[0168] The third combination consists of 23 sgRNAs. The number of sgRNAs in the second combination was reduced from 46 sgRNAs to 23 sgRNAs (only one of the two adjacent sgRNAs was retained). The sgRNAs used are: sgRNA1, sgRNA5, sgRNA9, sgRNA13, sgRNA17, sgRNA21, sgRNA25, sgRNA29, sgRNA33, sgRNA37, sgRNA41, sgRNA45, sgRNA49, sgRNA53, sgRNA57, sgRNA61, sgRNA65, sgRNA69, sgRNA73, sgRNA77, sgRNA81, sgRNA85 and sgRNA89.

[0169] The first combination of 91 sgRNAs was obtained by secondary amplification and transcription mixing. All 91 forward primers were divided into two groups (46 and 45). After mixing, every 46 or 45 forward primers were used together as forward primers and reverse primers to PCR amplify the plasmid template to obtain 46 or 45 sgDNA template fragments. The sgRNA in vitro transcription kit (Yinmao Shengye) was used to transcribe the 46 or 45 mixed sgDNAs obtained in the previous step as templates to obtain the corresponding 46 or 45 mixed sgRNAs. After two amplification and transcription experiments, all 91 mixed sgRNAs were obtained after mixing.

[0170] The 46 sgRNAs of the second combination and the 23 sgRNAs of the third combination were amplified and transcribed once. All 46 or 23 forward primers were mixed and used as forward and reverse primers to obtain 46 or 23 sgDNA template fragments from the plasmid template through a single PCR amplification reaction. The sgRNA in vitro transcription kit (Yinmao Shengye) was used to transcribe the 46 or 23 mixed sgDNAs obtained in the previous step as templates to obtain the corresponding 46 or 23 mixed sgRNAs.

[0171] 7.4 Editing and Binding of UdgX Protein to DNA Fragments

[0172] The UdgX-SSBE3 protein bound to the snap magnetic beads and the corresponding sgRNA combination (91 sgRNAs were added to 10uL, 46 sgRNAs were added to 5ul, or 23 sgRNAs were added to 3uL) were added to 0.5mL of reaction buffer (50mM Tris-HCl, pH8, 50mM NaCl, 1mM Na2EDTA, 1mM DTT, 25ug / ml BSA), reacted at 25°C for 5 minutes, and then 8ug of human salivary genomic DNA after double-stranding was added, and reacted at 37°C for 2 hours.

[0173] 7.5 Obtaining target DNA fragments

[0174] After reacting at 37°C for 2 hours, gently invert the centrifuge tube several times to resuspend the magnetic beads and perform magnetic separation. Remove the reaction solution and add 1 mL of buffer (50 mM Tris-HCl buffer, 50 mM NaCl, pH 8) to the centrifuge tube containing the magnetic beads. Gently invert the centrifuge tube several times to resuspend the magnetic beads and perform magnetic separation. Repeat three times. Finally, add 500 μL of buffer (50 mM Tris-HCl buffer, 50 mM NaCl, pH 8) and add proteinase K to the eluate of the UdgX protein-DNA fragment complex to a final concentration of 100 μg / mL. Treat at 50°C for 30 minutes to decrosslink the UdgX-SSBE3 protein-DNA fragment complex and obtain the target DNA fragment.

[0175] 7.6 Quantitative Analysis of Target DNA Fragments

[0176] The target DNA after cross-linking was quantified by q-PCR, and the target DNA was amplified from the cross-linked DNA using primers (the third combination was the target DNA corresponding to 23 sgRNAs). The results showed that UdgX-SSBE3 could specifically capture the target DNA fragment containing the target editing site in the human saliva genome, and the capture amount of the 23 target DNAs was 3.95*10-5 -5.85*10 -4 ng (using the third combination containing 23 sgRNAs for capture), 3.86*10 -5 -1.6*10 -3 ng (using the second combination containing 46 sgRNAs for capture) and 1.18*10 -4 -8.17*10 -4 ng (capture was performed using the first combination containing 91 sgRNAs) ( FIG15 ).

[0177] q-PCR results showed that the capture yield of target sites using the 23-sgRNA combination was significantly lower than that using the 46- or 91-sgRNA combinations. While the capture yields of target fragments using the 46-sgRNA combination and the 91-sgRNA combination were similar, the 91-sgRNA combination provided more uniform capture across all sites, facilitating next-generation sequencing.

[0178] 7.7 Next-generation sequencing of target DNA fragments

[0179] Using the cross-linked target DNA (captured by a combination of 46 or 91 sgRNAs) as a template, the VAHTS Universal DNA Library Prep Kit for Illumina V3 was used to construct a second-generation library. The constructed DNA library was sent to Biomark Biotechnology Co., Ltd. for second-generation sequencing. The sequencing results showed that the long target DNA (41KB) captured by the 46 sgRNA combination had three gaps after sequencing and could not capture the complete 41kb long fragment. The long target DNA (41KB) captured by the 91 sgRNA combination had no gaps after sequencing and could capture the complete 41kb long fragment. The coverage uniformity of the entire fragment was significantly better than that of the 46 sgRNA combination (Figure 16).

[0180] Example 8: Using a combinatorial library containing 142 sgRNAs to capture the entire exon sequence (total 67997 bp) of three childhood tumor-related genes (TRIM28, FBXW7, and NYNRIN) from human saliva genomic DNA samples

[0181] 8.1 Expression and purification of UdgX-SSBE3 protein (same as Example 1)

[0182] 8.2 Acquisition of Genomic DNA Samples (Same as Example 1)

[0183] 8.3 Obtaining sgRNA Fragments

[0184] Based on Example 7, 51 additional sgRNAs were designed using conventional design methods to capture the remaining 46 exon sequences of the three genes, excluding the 41 kb long fragment (for ease of presentation, the transcription templates corresponding to the 51 sgRNAs are represented by sgDNA92-sgDNA142). Therefore, there are a total of 142 sgRNAs used to capture all exon sequences of the three genes related to childhood tumors (a total of 47 exons, 67997 bp in length).

[0185] The corresponding forward primers were used as mixed primers and reverse primers to obtain template sgDNA fragments (sgDNA1-sgDNA142) corresponding to the target editing site from the plasmid template by PCR amplification. However, there were many 142 sgRNAs, so they were amplified and transcribed three times to obtain a mixture. All 142 forward primers were divided into three groups (46, 45, and 51). After mixing each group of forward primers, they were used together as forward primers and reverse primers to PCR amplify the plasmid template to obtain three groups of sgDNA template fragments. The sgRNA in vitro transcription kit (Yinmao Shengye) was used, and the three groups of mixed sgDNA obtained in the previous step were used as templates to transcribe the corresponding three groups of mixed sgRNAs. After three amplification and transcription experiments, all 142 mixed sgRNAs were obtained after mixing.

[0186] 8.4 Editing and Binding of UdgX Protein to DNA Fragments

[0187] The UdgX-SSBE3 protein bound to the snap magnetic beads and the corresponding sgRNA combination (15ul of 142 sgRNAs were mixed) were added to 0.5mL of reaction buffer (50mM Tris-HCl, pH8, 50mM NaCl, 1mM Na2EDTA, 1mM DTT, 25ug / ml BSA), reacted at 25°C for 5 minutes, and then 8ug of human salivary genomic DNA after double-stranding was added and reacted at 37°C for 2 hours.

[0188] 8.5 Obtaining the target DNA fragment (same as Example 1)

[0189] 8.6 Quantitative Analysis of Target DNA Fragments

[0190] The cross-linked target DNA was quantified by q-PCR, and primers were used to amplify a portion of the target DNA (the remaining target DNA captured by the 51 sgRNA combinations except for the 41KB long fragment) from the cross-linked DNA. The results showed that UdgX-SSBE3 can specifically capture the remaining exon fragments of three pediatric tumor-related genes in the human saliva genome, except for the 41KB long fragment. The capture amount of 51 newly added target DNA fragments was 6.17*10-5 -5.7*10 -4 ng (Figure 17).

[0191] 8.7 Next-generation Sequencing of Target DNA Fragments

[0192] Using the decrosslinked target DNA as a template, a second-generation library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 from Novozymes. The constructed DNA library was then sent to Biomike Biotechnology Co., Ltd. for next-generation sequencing. Sequencing results showed that the 142 sgRNA combinations could capture the exonic regions of three pediatric cancer-related genes in the human salivary genome. All exonic target fragments were specifically captured, while intronic regions were largely undetected (Figure 18).

[0193] A total of 67,997 base positions in the exon were fully covered, with a coverage depth ranging from 26 to 8,134 base positions and an average sequencing depth of 5,958.74 base positions. Fifty-three positions had a coverage depth of less than 30 base positions, and 237 positions had a coverage depth of less than 100 base positions (Figure 19).

[0194] Example 9: Using a combinatorial library containing 176 sgRNAs to capture the entire exon sequence (total 67997 bp) of three childhood tumor-related genes (TRIM28, FBXW7, and NYNRIN) from human saliva genomic DNA samples

[0195] Based on Example 8, 34 additional sgRNAs were designed to assist in capturing target sequences with low sequencing depth in the next-generation sequencing results of the sample captured in Example 2 (the transcription templates corresponding to the 34 sgRNAs were sgDNA143-sgDNA176). Thus, a total of 176 sgRNAs were designed to capture all exon sequences of three genes related to childhood tumors (a total of 47 exons, 67,997 bp in length).

[0196] 9.1 Expression and purification of UdgX-SSBE3 protein (same as Example 1)

[0197] 9.2 Acquisition of Genomic DNA Samples (Same as Example 1)

[0198] 9.3 Obtaining sgRNA Fragments

[0199] The corresponding forward primers and reverse primers were synthesized. The forward primers used were mixed primers and reverse primers. The template sgDNA fragments corresponding to the target editing site (sgDNA1-sgDNA176) were amplified from the plasmid template by PCR. Due to the large number of 176 sgRNAs, they were amplified and transcribed in four batches. All 142 forward primers were divided into four groups (46, 45, 43, and 42). After mixing, each group of forward primers was used as a forward primer and reverse primer to obtain four groups of sgDNA template fragments from the plasmid template by PCR amplification. The sgRNA in vitro transcription kit (Yinmao Shengye) was used to transcribe the four groups of mixed sgDNA obtained in the previous step as templates to obtain the corresponding four groups of mixed sgRNAs. After four amplification and transcription experiments, all 176 mixed sgRNAs were obtained after mixing.

[0200] 9.4 Editing and Binding of UdgX Protein to DNA Fragments

[0201] The UdgX-SSBE3 protein bound to the snap magnetic beads and the corresponding sgRNA combination (a total of 20ul after mixing 176 sgRNAs) were added to 0.5mL of reaction buffer (50mM Tris-HCl, pH8, 50mM NaCl, 1mM Na2EDTA, 1mM DTT, 25ug / ml BSA), reacted at 25°C for 5 minutes, and then 8ug of human salivary genomic DNA after double-stranding was added, and reacted at 37°C for 2 hours.

[0202] 9.5 Obtaining the target DNA fragment (same as Example 1)

[0203] 9.6 Quantitative Analysis of Target DNA Fragments

[0204] The cross-linked target DNA was quantified by q-PCR, and primers were used to amplify the target DNA with low coverage (the target DNA with low coverage mediated by 34 newly designed sgRNAs) from the cross-linked target DNA. The results showed that the capture of these target DNAs was improved by adding new sgRNAs to assist in capture. The capture amount of the 34 newly added target DNA fragments was 5.61*10 -4 -2.56*10 -3 ng (Figure 20).

[0205] 9.7 Next-generation Sequencing of Target DNA Fragments

[0206] Using the cross-linked target DNA as a template, a second-generation library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 from Novozymes. The constructed DNA library was then sent to Biomike Biotechnology Co., Ltd. for second-generation sequencing. Three biological replicates were performed, and all captured target DNA samples underwent second-generation sequencing. Sequencing results showed that the 176 sgRNA combinations could capture the exonic regions of three pediatric tumor-related genes in the human saliva genome. All exonic target fragments were specifically captured, while intronic regions were largely undetectable (Figure 21).

[0207] The exon contains a total of 67,997 base positions, all of which were fully covered, with an average coverage depth of 587-8018 and an average sequencing depth of 5958.74 (Figure 22). Target fragments captured using this method were subjected to next-generation sequencing, and the corresponding sequencing data had a Fold80 value of 1.348, 0.2*mean>99.3%, 0.5*mean>92.1%, and 0.5_2x_mean>92.1%, with 30X coverage of 100%. This capture performance is comparable to sequencing parameters for commercially available probe hybridization capture methods.

[0208] 9.8 Analysis of off-target sites of UdgX-BE3 protein

[0209] The sequencing data of the three samples were aligned with the human whole genome sequence as the reference genome, and there were 573 sites with a sequencing depth of more than 400 reads (average sequencing depth of 1 KB fragments per site) (Figure 23).

[0210] There are 573 sites with a sequencing depth greater than 400, of which 408 are located on chromosomes at non-target sites. For sites located on non-target chromosomes, the length distribution statistics were made for sites with a sequencing depth greater than 1000. A total of 175 sites had a sequencing depth greater than 1000, with an average length of 1072.68bp. Of the 175 sites with a sequencing depth greater than 1000, 76 had an actual length of less than 500bp after splicing of the site sequences, and the actual length of the remaining 99 sites was greater than 500bp. For fragments greater than 500bp in the sequencing data, this may be due to off-target sites of the base editor in the UdgX-SSBE3 fusion protein (Figure 24).

Claims

1. A fusion protein composed of a cytosine base editor and a uracil binding protein; More preferably, the uracil binding protein is UdgX, specifically, its UdgX includes the sequence of motif A: GEQPG and motif B: HPSSLL, and contains the conserved region KRRIH; more specifically, the uracil binding protein UdgX protein is from M.smegmatis, M.avium, R.imtechensis, M.haemophilum, Rhodococcusspp, Streptomycescoelicolor, Gordonianamibiense, Bradyrhizobiumjaponicum and Nocardia farcidia; more preferably, its amino acid sequence is shown in SEQ ID No.22; The cytosine base editor is a base editor that can produce uracil U after base editing, and specifically the cytosine base editor is a BE3 editor (such as VQR-BE3, VRER-BE3, EQR-BE3, etc. that recognize different pam types), an AID editor, or an optimized SSBE3 editor thereof; The cytosine base editor and the uracil binding protein are connected without or through a linker, and the specific amino acid sequence of the linker is shown in SEQ ID NO:

24.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID No.

1.

3. The fusion protein according to claim 1 or 2, characterized in that The fusion protein also has a purification tag, which facilitates easy separation and / or purification; specifically, the purification tag includes Snap-tag, His-tag, Flag-tag, MBP-tag, and biotin tags for protein purification.

4. A polynucleotide encoding the fusion protein according to any one of claims 1 to 3, an expression vector and a recombinant host cell, particularly, the polynucleotide sequence is as shown in SEQ ID No.

2.

5. A method for capturing target DNA from a sample, the method comprising: 1) contacting a sample containing a target sequence DNA having a PAM and containing cytosine C at a specific position with its targeting sgRNA and the fusion protein according to any one of claims 1 to 3, such as mixing, to obtain a fusion protein-target DNA complex; 2) recovering the enriched target sequence DNA, preferably, wherein the target sequence DNA is double-stranded DNA or single-stranded DNA or a mixture of the two; Preferably, step 2) further comprises binding the fusion protein-target DNA complex to an affinity matrix, preferably magnetic beads, more preferably Snap magnetic beads; The specific position containing cytosine C refers to the 3rd to 25th base upstream from the 5' end of the PAM site, preferably the 5th to 20th base, and more preferably the 11th to 17th base.

6. The method according to claim 5, characterized in that The sample is from plants, animals and / or microorganisms, such as bacteria, archaea, protists, fungi, mammals, amphibians, birds, reptiles, insects and / or invertebrates, such as humans, more preferably, the sample is from blood, serum, serous fluid, plasma, lymph, urine, cerebrospinal fluid, saliva, mucosal secretions of secretory tissues and organs, vaginal secretions, milk, tears and / or ascites of the subject; Specifically, the target sequence DNA is exogenous DNA relative to the subject, such as DNA from pathogenic microorganisms, such as viruses, bacteria and / or fungi, or endogenous DNA of the subject, such as DNA from the subject containing a gene mutation, such as DNA containing a pathogenic gene mutation. In addition, preferably, the sample comprises a genomic DNA sample, a cell-free DNA sample, an environmental genomic sample and / or a mixed genomic DNA sample.

7. The method according to claim 5, characterized in that Wherein in step 1) of the method, a sample containing the target sequence DNA is contacted with the fusion protein and a combination of different types of targeting sgRNAs to obtain a uracil binding protein-target DNA complex; the sgRNA combination is a mixed sgRNA library targeting multiple sites; Specifically, the concentrations of each sgRNA were 0.1ng / ul-1ng / uL, 0.2ng / ul-2ng / uL, and 2ng / ul-20ng / uL; Preferably, the sgRNA combination used is designed in such a way that at least one sgRNA is designed for each 1KB target sequence captured; the more sgRNAs are used to capture each 1KB target sequence, the higher the capture amount thereof in the target fragments captured in batches; In the simultaneous capture of multiple targets, the distribution density of sgRNA is increased; optionally, for target sites with low coverage of second-generation sequencing, multiple corresponding sgRNAs are designed and invested to increase the capture amount of target fragments; Further preferably, the library of the sgRNA combination is obtained by using batch synthetic sgRNA or batch forward mixed primers and specific reverse primers to amplify and obtain the corresponding sgDNA combination library, and then detecting in a high-throughput manner by batch transcription to obtain the library of the sgRNA combination; Also preferably, in step 1) of the method, for simultaneously capturing multiple dispersed target fragments or capturing a long target fragment that needs to be divided into multiple smaller fragments and then spliced, the library of sgRNA combinations in the capture reaction system is mixed and put into batches.

8. A method for detecting a target DNA, the method comprising the steps of capturing the target DNA by the method according to any one of claims 5 to 7, and detecting whether the target DNA exists; Preferably, the step of capturing the target DNA further includes a step of amplifying the captured target DNA after obtaining the captured target DNA.

9. Use of the fusion protein according to any one of claims 1 to 3 for enriching and / or detecting a DNA containing a target sequence having a PAM and containing cytosine C at a specific position from a sample.

10. The fusion protein according to claims 1-3, and the use of the method according to any one of claims 5-9 in detecting off-target sites, or in enriching and / or detecting natural uracil in a sample, wherein in the application, after targeted capture sequencing by the method, the captured sample information is obtained by second-generation sequencing to determine the off-target detection site.

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