CN110158157B

CN110158157B - Method for synthesizing DNA library with fixed length and specific terminal sequence based on template material

Info

Publication number: CN110158157B
Application number: CN201810150519.6A
Authority: CN
Inventors: 贾俊岭; 潘辰; 李然
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2018-02-13
Filing date: 2018-02-13
Publication date: 2021-02-02
Anticipated expiration: 2038-02-13
Also published as: CN110158157A

Abstract

The invention relates to the field of biology, and particularly provides a method for synthesizing a DNA library with fixed length and specific terminal sequence based on template materials, which comprises the following steps: obtaining fragmented double-stranded DNA; DNA end repair and 3' end tailing; linkage of the 3' Biotin modified a1 linker; purifying a connection product; DNA is fixed on a solid phase with streptavidin; denaturing the double-stranded DNA into single strands; hybridizing the primer 1; extending bases with dNTPs having reversible chemical modification at the 3' hydroxyl of a deoxyribose group; recovering the hydroxyl at the 3' end of the pentose; a fixed length DNA extension; flattening the 5' end; selecting a terminal sequence; removing a terminal sequence; ligation of linkers for molecular cloning. The method can conveniently construct the region fixed length and the specific terminal sequence library which have no reference genome and high sequence variability, and greatly reduces the construction cost of the library.

Description

Method for synthesizing DNA library with fixed length and specific terminal sequence based on template material

Technical Field

The invention relates to the field of synthetic biology, in particular to a preparation method of a DNA library with fixed length and specific terminal sequence based on template material synthesis.

Background

A set of molecular biological mechanisms for regulation and control are arranged behind the life process and the biological behavior, and the rule of the molecular mechanisms is the central rule, namely the rule of information transmission among DNA, RNA and protein. In the last 30 years, the means for studying biological mechanisms mainly include forward genetics and reverse genetics, wherein the forward genetics are based on phenomena to search key molecules and mechanisms, and the reverse genetics are based on molecules to disclose mechanisms and search corresponding biological processes by controlling the molecules. For forward genetic research, a large technical means is genetic screening, and initially, aiming at a cell mechanism, unbiased screening is mostly adopted, while with the advent of the RNA interference technology, crispr (clustered regulated Short Palindromic repeat) technology, the screening of a library pool also becomes one of important gene screening modes.

The CRISPR system is a part of the bacteriophage-resistant immunity of bacteria, with which about 40% of bacteria and 90% of archaea resist bacteriophage infection. When the bacteria are attacked by phage or plasmid, a small part of the bacteria can integrate the DNA of the infectious source into the DNA cluster of CRISPR, and when infected again by the same pathogen, transcription of this region can be initiated, and can form a complex with Cas9 protein, clearing the DNA of the pathogen.

In 2013 or so, Zhang Feng et al achieved genomic DNA editing in mammals based on the immune defense system of bacteria. The system mainly comprises 2 components, wherein one component is a guide RNA for identifying genomic DNA according to base complementary pairing, and the other component is an endonuclease Cas9 recruited by the guide RNA, when the components and the genomic DNA form a complex, double-strand breaks of DNA can be caused, and insertion or deletion mutation can be generated at the breaks due to non-homologous end repair mechanisms of mammals, so that the genomic DNA sequence is edited. At the beginning of the development of the technology, gene knockout or knock-in is mainly realized in a cell line or a living body as a tool for manipulating genomic DNA. Due to the simplicity of the guide RNA design, the technology is changed into a powerful genomics research tool by a plurality of groups, for example, the technology is utilized by Zhang Feng to screen out melanoma drug resistance related genes NF1, NF2 and CUL 3. Due to the ease of manipulation of the guide RNA for genes, scientists have also designed Cas9 protein as an activated or suppressed version, conveniently achieving gene activation or suppression. Mammalian cells are primarily cis-regulated compared to lower organisms such as Drosophila. Zhang Feng et al studied the function of the non-coding region DNA around the CUL3 gene using CRISPR technique, and they found that the binding site of transcription factors such as CTCF, YY1 had a regulatory effect on CUL 3. The CRISPR technology of anyone et al studied cis-acting elements around POU5F1 gene in human embryonic stem cells, and they found that cis-acting elements regulating POU5F1 exist inside other genes, while when the system of zhangfeng et al studied lncRNA, it was found that activating lncRNA transcription on genome could produce cellular resistance, but lncRNA itself did not cause resistance, suggesting that multi-layer annotation may be required for genome annotation, not just as one state. Klann TS et al found that different cis-acting elements were required to regulate gene transcription in different cells, suggesting that the genomic DNA annotation of the present invention needs to be performed in different tissue cells. In view of the above, the study of genomic DNA functions using omics is becoming an important field.

WO2015065964 discloses a method for designing sgrnas against a reference genome using bioinformatics tools, and synthesizing a sgRNA library using a pool of primers. The price of the existing primer pool synthesis method is high, and if tens of millions of sgRNAs are synthesized, the cost is very high; meanwhile, the strategy must depend on a reference genome, so that species without the reference sequence genome, such as intestinal microorganisms or unknown species, cannot be subjected to CRISPR library construction; meanwhile, sgrnas designed from reference genomes may not function in individuals without sequencing, having nucleotide sequences that differ between individuals of the same species. The cost is reduced, and meanwhile, the construction of a gRNA library by using natural nucleic acid as a material is a great technical development direction at present.

WO 2017081097A 1 discloses a method for constructing a gRNA library of natural nucleic acid by using a 20bp short fragment library obtained by using a type II S restriction endonuclease, but the method is complex in operation process, and sgRNA diversity is greatly lost due to the fact that a plurality of enzyme cutting sites need to be fixed, so that a simpler and cheaper method is needed for preparing the gRNA library.

Therefore, there is a need for further research and development of new, simpler, efficient methods for the preparation of gRNA libraries.

Disclosure of Invention

Aiming at the current technical situation, the method can convert the naturally-occurring DNA or nucleic acid into the sgRNA library, and the method does not need to design the sgRNA by using the reference sequence, but directly converts the natural DNA into DNA with fixed length, such as 19-24bp, and the fragments with fixed length are derived from DNA materials, so that the condition that the design of the reference sequence is inaccurate does not exist.

The invention provides a sequence selection method of a specific end and is used together with DNA (deoxyribonucleic acid) with a fixed length, so that a sgRNA library can be obtained.

The invention relates to a preparation method of a DNA library with fixed length and specific terminal sequence based on template material synthesis, which comprises the following steps:

1) primer hybridization: taking a single-stranded DNA with a linker A1 immobilized on a stationary phase with streptavidin,

in one embodiment, the linker a1 in step 1) is a peptide having the nucleotide sequence shown in SEQ ID No.1 and/or SEQ ID No.2, or the linker a1 is CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, (Gal1,10), TEF1, GDS, ADH1, CaMV35s, Ubi, H1, U6; or a 3' terminal sequence of T7, T7lac, Sp6, araBAD, trp, lac, Ptac, or Pl;

the A1 linker is used to clone the final product into the promoter region of the cloning vector, and thus this sequence is derived from the promoter region sequence of the cloning vector, typically the sequence at the 3 'end of the promoter region, and may extend the sequence at the 5' end of the A1 linker; as one of the embodiments, cloning vector promoter sequences for eukaryotic expression include CMV, EF1a, SV40, PGK1, Ubc, human beta actin, CAG, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, (Gal1,10), TEF1, GDS, ADH1, CaMV35s, Ubi, H1, or U6; as another embodiment, cloning vector promoters for prokaryotic expression include T7, T7lac, Sp6, araBAD, trp, lac, Ptac, pL.

As one embodiment, it is preferred that the linker A1 in step 1) is a peptide having the nucleotide sequence shown in SEQ ID NO.1 and/or SEQ ID NO. 2;

as one embodiment, the primer 1 in step 1) is SEQ ID NO: 11;

wherein, the primer 1and the primer 2 are respectively complementary sequences to A1-R and A1-F;

as one embodiment, the gradient annealing in the step 1) is 85 ℃ for 30s and 65 ℃ for 1min, and the temperature is slowly reduced to 40 ℃ at 0.1 ℃ per second and is 5min at 40 ℃;

as one embodiment, the DNA hybridization buffer solution in the step 1) is a5 XSSC buffer, which is a mixed solution of (0.75M sodium chloride, 0.075M sodium acetate, pH 7.0)/0.1% Tween 20.

2) Base elongation: then adding DNA polymerase and dNTPs with reversible chemical modification at the 3 'end to prolong the base, and then recovering the hydroxyl at the 3' end;

as one embodiment, the DNA polymerase in step 2) is 9 ° N (D141A/E143A/a485L) DNA polymerase;

as one embodiment, the dNTPs with reversible chemical modification in step 2) are 3' -O-azidomethyl-dNTPs, 3' -O-allyl-dNTPs or 3' -O-allyloxycarbonyn-dNTPs;

as one embodiment, the step 2) is performed to restore the 3' terminal hydroxyl group using tris (2-carboxyacetic acid) phosphine hydrochloride, TECP or DTT;

as one embodiment, the step 2) further comprises adding extension buffer solution when extending the base, incubating at 65 degrees for 2min, washing the magnetic beads with washing buffer solution 1 for 2 times, then incubating at 65 degrees with 3' hydroxyl-terminated recovery buffer solution for 1min, washing with washing buffer solution 2 for 2 times, and repeating the above processes if necessary;

as one embodiment, the extension buffer in the step 2) is a mixed solution of 1. mu.M 3' -O-azidomethyl-dNTPs mix, 0.015. mu.g/ml 9 ℃ N DNA polymerase,50mM Tris pH 9.0,50mM NaCl,6mM MgSO4,1mM EDTA and 0.05% Tween 20;

as one embodiment, the washing buffer 1 in the step 2) is a mixed solution of 50mM Tris pH 9.0,50mM NaCl,1mM EDTA and 0.05% Tween 20;

as one embodiment, the 3' -terminal hydroxyl group-recovering buffer in the step 2) is a mixed solution of 100mM TCEP (Tris- (2-Carboxyethyl) phosphine, Tris (2-Carboxyethyl) phosphine hydrochloride), 100mM Tris pH 9.0,100mM NaCl,50mM sodium ascorbate, and 0.05% Tween 20;

as one embodiment, the washing buffer 2 in the step 2) is a mixed solution of 100mM Tris pH 9.0,100mM NaCl, and 0.05% Tween 20;

3) fixed length DNA extension: repeating the cycle of the step 2) for 15-100 times, preferably 20-30 pb, and further preferably 20-23 pb;

4) flattening the tail end: the 5' -end was removed by adding a single-stranded nuclease,

as one embodiment, the single strand nuclease in step 4) is mungbean acid nuclease or S1 nuclease CEL I; as one of the further embodiments, mung bean nuclease is preferably used;

5) selection of terminal sequences: the selection of the terminal sequence has been carried out by adding DNA ligase and linker B1; or replacing to add base for extension in the circulation corresponding to the position of the sequence to be selected, and purifying DNA with complete length by polyacrylamide gel electrophoresis to complete end selection;

as one embodiment, said DNA ligase in said step 5) is T4DNA ligase or blunt-end/TA ligase;

as one embodiment, the linker B1 in step 5) comprises a sequence of AscI, Acc65I, AccB1I, ApaI, Asp718I, AspS9I, AvaII, BamHI, BanI, BbeI, Bme18, 18I, BmgT120I, BmiI, BshFI, BshNI, BsnI, Bsp120I, BspLI, BstEII, BstPI, BsuRI, BtsCI, Eco91I, EheI, FseI, HaeIII, KasI, KpnI, PhoI, sanddi, SfoI, SgsI, or ssdi cleavage sites; preferably, linker B1 is the nucleotide sequence shown in SEQ ID NO.3 and/or SEQ ID NO.4, or the sequence of AsiSI, BsePI, BssHII, NotI, HhaI, PauI, SfaAI;

as an embodiment, optionally, the step 5) further comprises selecting the terminal sequence after selecting the terminal sequence by using a B1 linker or selecting the terminal sequence again by using a B2 linker, wherein the linker B2 is the nucleotide sequence shown in SEQ ID NO.5 and/or SEQ ID NO. 6.

6) Removal of terminal sequences: adding DNA ligase and a linker B3, and removing a terminal sequence;

in the method of the present invention, as one of the embodiments, the DNA ligase is T4DNA ligase, or blunt end/TA ligase;

in the method of the present invention, as one embodiment, the linker B3 is a sequence comprising Type II S restriction endonuclease, preferably a sequence comprising BbsI, AcuI, AlwI, BaeI, BccI, bcei, BbvI, BcoDI, BfuAI, BpuEI, BsaXI, BseRI, BsgI, BsmBI, BsmFI, bsmmi, BspCNI, BspMI, BspQI, BtgZI, EarI, Ecil, FauI, HgaI, HphI, HpyAV, MmeI, MnII, SapI, or nisattype II S restriction endonuclease; further preferably, the linker B3 is the nucleotide sequence of SEQ ID NO.7 and/or SEQ ID NO. 8;

7) ligation of cloning adaptor: adding DNA ligase and a joint B4 to carry out the connection of the cloning joint; in the method of the present invention, as one embodiment, the DNA ligase is T4DNA ligase, blunt end/TA ligase;

in the method of the present invention, as one embodiment, the linker B4 comprises: 1) cas9 knockdown of the relevant chimeric RNA sequence (addgene: #52961, LentiCRISPR V2); 2) cas9 activates the relevant chimeric RNA sequence (addgene: # 73797); 3) cpf1 (addgene: #84739 and # 84740).

In the method of the present invention, as one embodiment, in the step 2), 1 base extension is performed using dNTPs having reversible chemical modification of the hydroxyl group at the 3' end of the deoxyribose group: adding dNTPs containing DNA polymerase and reversible chemical modification at the 3' end into the magnetic bead DNA compound in the step 1); the method of the present invention is preferably O-azidomethyl at the 3 'end, but the present invention can also adopt other dNTPs with 3' hydroxyl of deoxyribose group and reversible chemical modification to replace 3'-O-azidomethyl-dNTPs used by the method, and the present invention includes but is not limited to 3' -O-azidomethyl-dNTPs, 3 '-O-allyl-dNTPs or 3' -O-allyloxy-borony-dNTPs.

dNTPs with reversible chemical modification of the hydroxyl group at the 3' end have 2 characteristics, and a) can be doped into an extended chain by DNA polymerase; b) the modification can be efficiently cleaved off and 3' restored to hydroxyl; thus, reversible end modifications having both of the above-described characteristics can be used in the method of the present invention.

In the method of the present invention, as one embodiment, the step 3) is a step of extending the fixed-length DNA; the extension of base pairs is carried out by those skilled in the art according to actual needs, such as but not limited to 15-100 pb, for example, 23bp extension is needed, and the step 2) is repeated twenty times. The invention repeats step 2) to carry out multi-cycle reversible end reaction, and then obtains a sequence with fixed length through the single-stranded nuclease reaction of step 3), wherein the length of the sequence can be 15bp to 100 bp; via step 3), an extended DNA single strand may be obtained via a DNA denaturation step such as heat denaturation or other denaturing amount, and then linked to PAM using a DNA single strand ligase to select the relevant single stranded oligonucleotide SEQ ID NO:4, obtaining the DNA structure in the step 5) through PCR reaction.

In the method of the present invention, as one embodiment, the step 5) is optional, wherein, during the step 3) cycle, specific 3' -O-azidomethyl-dNTPs are added in the 1 st cycle reaction, and after the sequence extension with specific characteristics is completed, the step 5) operation is not required;

adding DNA ligase and a B1 adaptor in the step 5), combining with the tail end of the extended DNA to form an enzyme cutting site, and selecting the required fragment by using the enzyme cutting site. The B1 joint comprises 1 enzyme cutting site part, and can form 1 enzyme cutting site together with the terminal nucleic acid extended in the step 2), and a sequence with a specific terminal can be selected after being cut by a DNA endonuclease.

In the method, in the step 6), DNA ligase and a B3 adaptor are added for later PCR amplification of a ligation product; wherein the B3 adaptor contains 1 type 2S restriction enzyme site, and can cut DNA and excise needed number of bases.

In the method of the present invention, in the step 7), a DNA ligase and a B4 linker are added, and the step 7) is used for connecting sequences required for molecular cloning, and fragments can be cloned on a vector for testing by using an existing cloning technique such as a conventional cloning technique or a seamless connection technique; the sequence can be selected by those skilled in the art according to the final vector and general knowledge in the art, and the vectors of the present invention include, but are not limited to, chimeric RNA sequence, LentiCRISPR V2 or Lenti-guide sequence, among other vectors available for commercialization at present.

In the method of the present invention, as one embodiment, the single-stranded DNA having the linker a1 immobilized on the stationary phase having streptavidin in the step 1) can be prepared by a method commonly used in the art, for example, the double-stranded DNA can be first denatured into single strands by heat denaturation or a denaturing agent sodium hydroxide, and the solution can be returned to 1M sodium chloride environment for streptavidin and biological affinity reaction, thereby immobilizing the DNA having the a1 linker on the stationary phase.

As one embodiment, the present invention includes, but is not limited to, the following method:

1-1) acquisition of fragmented double-stranded DNA: double-stranded DNA can be reverse transcribed using either ultrasound or fragmented RNA to obtain fragments of 10bp to 40 Kbp;

1-2) DNA end repair and 3' end tailing, namely mixing the DNA obtained in the step (1) with DNA polymerase to carry out DNA 3' end flattening, and adding A or G to the 3' end;

in the method of the present invention, as one embodiment, the DNA polymerase includes: 3' end with A tail, using a klenow fragment (3' → 5' exo)^-) (ii) a A G-tail was added to the 3' end, and Taq DNA polymerase was used.

1-3) ligation of 3' -Biotin modified A1 linker: mixing the product of the step (2) with library ligase, and adding an A1 linker; the ligase is blunt end/TA ligase;

1-4) purification of the ligation product: double-stranded DNA was purified using 1 Xvolume of Ampure XP beads, or DNA was recovered using ethanol or isopropanol precipitation or DNA silica membrane column purification kits.

1-5) binding of DNA to a solid phase: mixing the DNA obtained in the step 1-4) with an isovolumetric stationary phase with the concatemer, and incubating at room temperature;

in the method of the present invention, as one embodiment, the solid phase in step 1-5) comprises 2X B & W buffer resuspended T1 magnetic beads, Controlled porous Glass (Controlled Pore Glass), silica gel (silica gel), porous resin (Pore resin), microarray slides (Glass slides), silicon-based chips (silica chips), or nylon membrane (nylon membrane); in the method of the present invention, as one embodiment, the incubation time at room temperature includes, but is not limited to, 15 min.

1-6) denaturation of double-stranded DNA into single strands: modifying DNA on the magnetic beads with 0.1N NaOH to obtain single strands;

in the method of the present invention, as one of the embodiments, the denaturation in the step 1-6) includes thermal denaturation or denaturation using a DNA denaturant, and the denaturant is sodium hydroxide, formamide or urea; sodium hydroxide is preferred.

In the method of the present invention, as one embodiment, the double-stranded DNA in step 1-1) may be a nucleic acid sample of any origin, including genomic DNA of any species, cDNA obtained by reverse transcription of RNA, DNA library obtained by targeted capture technology, DNA obtained by chromatin co-immunoprecipitation, and the like, DNA clones generated from biological genomic DNA, such as gene cDNA clones, bacterial artificial chromosome clones, yeast artificial chromosome clones, and the fragment size thereof is in the range of 10bp to 40 Kbp.

In the method of the present invention, as one embodiment, in the step 1-2), the DNA polymerase may flatten the end of DNA3 'and add a or G to the 3' end; as one embodiment, the DNA polymerase includes: when the A tail is added to the ' end of DNA3, the Klenow fragment (3' → 5' exo) is used^-) (ii) a When the 3' end of the DNA is added with a G tail, Taq DNA polymerase is used; as one embodiment, if Taq DNA polymerase is used for adding G to the 3 'end, T needs to be changed to G at the 3' end of the forward primer of the A1 adaptor;

the DNA is connected with an A1 linker to prepare for the subsequent reaction of accessing dNTPs with reversible chemical modification at the 3'; meanwhile, the linker A1 comprises a part of the sequence for molecular cloning, and if the vector is changed, the part of the sequence of the linker can be adaptively designed according to the vector, wherein the change of the A1 is mainly changed according to the target vector of cloning.

The DNA library produced by the method of the present invention can be cloned into a DNA vector, and according to the currently mainstream cloning method of the cloning library such as the seamless cloning method (seamless cloning method), if designed into a seamless cloning library, the sequence of A1 should be the same as the sequence of the assembly arm required for assembly, and one side is usually a promoter sequence, for example, A1 can be changed into the 3' end sequence of mU6 or T7 promoter; in the traditional cloning method, restriction endonuclease is used for preparing a library, A1 needs to contain a vector sequence to a restriction enzyme cutting site.

In the method of the present invention, as one embodiment, in the steps 1-4), the other PCR purification reagents include, but are not limited to, a DNA purification kit, and currently, DNA purification methods mainly used in laboratories include ethanol or isopropanol precipitation, silica gel column purification, and magnetic nucleic acid bead purification; nucleic acid magnetic bead purification methods are preferred, including, for example, but not limited to, Beckman brand Ampure XP magnetic beads.

In the method of the present invention, as one embodiment, in the step 1-5), when 2X B & W buffer is used, DNA is mixed with an equal volume of 2X B & W buffer resuspended T1 magnetic beads, and incubated at room temperature for 15 min.

As one embodiment, in the steps 1-5), other major solid phases for DNA single nucleotide chain synthesis include, but are not limited to, for example, Controlled Pore Glass, silica gel, porous resin, or Glass slides or silicon chips or nylon membrane used in a method for synthesizing DNA single nucleotide chains on a chip, wherein DNA single nucleotide chains of a specific sequence may be first immobilized on the surface thereof for hybridizing a DNA library, and before synthesizing DNA of a fixed length, the DNA may be hybridized by DNA single strand denaturation and annealing, i.e., the DNA single nucleotide chains may be first immobilized on the surface thereof; or by using DNA library with special chemical modification such as biotin modification, and directly fixing DNA to streptavidin resin or magnetic beads, and these materials are characterized by using a pair of molecules with strong interaction such as covalent binding, and other molecules with covalent bond interaction are modified, and can be used for synthesizing DNA library by using the method of the present invention.

In the method of the present invention, the immobilization of the DNA on the stationary phase enables efficient and rapid switching of the DNA reacted in the previous step into a solution, particularly in the step of DNA extension of a fixed length.

In the method of the present invention, as one embodiment, in the steps 1 to 6), the denaturation is performed by heat, or by using a DNA denaturing agent such as sodium hydroxide, formamide, or urea; sodium hydroxide is preferred.

As one embodiment, the present invention denatures DNA and then hybridizes with a primer to provide a DNA structure for reversible end reaction, which can achieve extension of the fixed length of the extended strand by incorporating dNTPs with reversible modification of the 3 'end into the 3' end of the primer using DNA polymerase, or can be formed using other methods, such as designing primer 1 to have a biotin-modified form, followed by heat denaturation annealing with the library and immobilization onto a solid phase.

In one embodiment of the method of the present invention, the fixed length is 15 to 100bp, preferably 20 to 30pb, and more preferably 20 to 23pb, which may be, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 pb. It will be understood by those skilled in the art that the fixed length means that the resulting DNA samples all have the same length, for example, all 20, 21, 22 or 23bp in length; the fixed length sequences may be the same base or different bases; the bases are the same or different, depending primarily on the template strand, if the bases are the same as the template strand bases, if the template has only one base, then the bases in the resulting sequence are the same; if there are multiple templates, the bases of the products are not identical.

In the method of the present invention, as one embodiment, the terminal specific sequence is a DNA library or a target DNA sample selected by using the library of the present invention has an NGG, NAG or NAA sequence at the terminal; as one embodiment, SpCas 93' NGG is preferred; SpCas9VRER:3' NGCG; SpCas9EQR:3' NGAG; SpCas9VQR is 3' NGAN or NGNG; saca 9:3' NNGRRT or NNGRR (N); AsCpf1and LbCpf1:5' TTTV; AsCpf1RR:5' TYCV; LbCpf1RR 5' TYCV; AsCpf1RVR:5' TATV; neisseria Meningitidis (NM):3' NNGATT; streptococcus Thermophilus (ST):3' NNAGAAW; or Treponema Dentanola (TD):3' NAAAAC.

As one embodiment, the method of the present invention can be used not only for the construction of a fixed length DNA of a criprpr vector, but also for the construction of an shRNA library.

In a second aspect, the present invention provides a method for sequence selection, the method comprising: in the method, only specific dNTP is doped during terminal sequence synthesis, the length of a target sequence is the target length, but the length of a non-target sequence is shorter than the length of the target sequence, the target length DNA is distinguished from the non-target length DNA by gel electrophoresis, and the target length DNA is purified, namely the screening of the specific sequence is completed.

The method can conveniently construct the fixed length and the specific terminal sequence library of the region without the reference genome and with high sequence variability, and simultaneously, the method can greatly reduce the construction cost of the library. The present invention proves the method of synthesizing library with fixed length and specific terminal sequence based on template strand, the DNA library synthesized by the method has the sequence structure as shown in figure 2-D, the middle part is 20bp sequence from initial DNA material, the fixed length of the library means that the length of the 20bp is fixed, the terminal is specific sequence, that is, before obtaining 20bp DNA in figure 2-D, the sequence following 20bp is shown in figure 2-A, the result shown in figure 2-C can be obtained by 2 methods provided by the present patent for screening, according to the left-to-right direction in the figure, the sequence on the right side of 20bp is NGG, the sequence on the terminal is NGG sequence.

Drawings

FIG. 1: the invention constructs a flow chart of sgRNA library construction;

FIG. 2: the DNA sequence of the PCR product after the B1-B4 joint is connected in the embodiment 1 of the invention;

FIG. 3: the results obtained in the end sequence selection and excision steps of the present invention are shown in example 1 by DNA agarose gel electrophoresis. A. After 23 times of 1bp extension and 3' hydroxyl recovery of the template DNA, a band of 144bp appears in the 5 th lane, and the sequence is shown in FIG. 2-A. B. After AscI digestion and ligation with B2 linker, a 142bp band appeared in lane 4, and its sequence is shown in FIG. 2-B. C. The DNA in Panel B was digested with AscI, resulting in a band of about 81bp in lane 5. D. The 4 th lane generated a 141bp band by BbsI cleavage to remove the terminal base, 5' end filling up and ligation of B4 adaptor, and the DNA sequence is shown in FIG. 2-D. D. The DNA in the third lane is distributed about 59 bp-60 bp by the amplification of the short primer and the electrophoresis of the modified polyacrylamide;

FIG. 4: the invention in the embodiment 1 generates a representative diagram of the distribution of the sequences in the 20bp sequence library in the mouse genome; the green part is the distribution of the starting DNA material-mouse embryonic stem cell H3K4me3 chromatin co-immunoprecipitated DNA in the mouse genome, and the red part is the representative distribution map of the 20bp library generated by the starting material through the operation steps of the invention in the mouse genome;

FIG. 5: the method of the embodiment 1 of the invention synthesizes a comparison chart of the conditions of 20bp sgRNA library and starting DNA in the mouse embryonic stem cell H3K4me3 area according to the template strand: FIG. A shows the DNA alignment of the starting material of example 1 to a murine embryonic stem cell H3K4me3, and FIG. C shows the alignment of the library obtained in example 1 to a murine embryonic stem cell H3K4me 3; panel B is the case of example 1 where the DNA of the starting material immediately follows the NGG sequence, and panel D is the proportion of the library obtained in example 1 immediately following the NGG sequence;

FIG. 6: the comparative figure of the condition of synthesizing 20bp sgRNA library of the human embryonic stem cell H3K4me3 area and the starting DNA according to the template strand in the method of the embodiment 2 of the invention is as follows: FIG. A shows the DNA alignment of the starting material of example 2 to human embryonic stem cells H3K4me3, and FIG. C shows the alignment of the library obtained in example 2 to human embryonic stem cells H3K4me 3; panel B is the case of example 2 where the DNA of the starting material immediately follows the NGG sequence, and panel D is the proportion of the library obtained in example 2 immediately following the NGG sequence;

FIG. 7: the comparison of the synthetic library of the method of example 2 of the present invention and the synthetic library of the primer pool is shown in the following figure: panel A is a proportion of DNA of each length obtained by synthesis in example 2, panel B is a proportion of DNA of 19 and 20bp immediately followed by NGG sequence in example 2, panel C is a proportion of DNA of each length obtained by synthesis in a commercial primer pool, and panel D is a proportion of DNA of 20bp immediately followed by NGG sequence obtained by synthesis in a commercial primer pool.

Detailed Description

The present invention will be further described with reference to the following examples or test examples, but the present invention is not limited thereto.

In the following examples, 20bp libraries were synthesized using mouse embryonic stem cell (example 1) and human embryonic stem cell (example 2) H3K4me3 modified region DNAs as starting materials, and examples 1and 2 were constructed according to the following procedure, except that the starting materials were different. These libraries are all 20bp long, but are not intended to limit in any way the utility of the invention to the synthesis of libraries of other lengths. Wherein FIGS. 1and 2 are process flow diagrams for library construction of the present invention, wherein the reagents and materials used in examples 1and 2 are as follows:

firstly, a reagent:

1. example 1 starting material: mouse embryonic stem cell H3K4me3 chromatin co-immunoprecipitation DNA

2. Example 2 starting material: human embryonic stem cell H3K4me3 chromatin co-immunoprecipitation DNA

NEBNext ultrafast end repair/dA tail addition Module, NEB, E7442

NEBNext ultra-fast connection module, NEB, E7445;

5.Ampure XP beads,Beckman，A63881；

6.Dynabeads^TMt1 magnetic bead, inteviety, 65602;

7.2 Xbind and wash buffer;

8. nuclease-free water, Ambion, 10977023;

9.1M sodium hydroxide, Sigma, S2770-100 ML;

10. a hybridization buffer;

11. an extension buffer;

12.3' hydroxy recovery buffer;

13. washing buffer solution 1;

14. washing buffer solution 2;

15.10X MBN buffer, NEB, M0250;

16.MBN(10U/μL),NEB,M0250；

17.10X T4DNA ligation buffer, NEB, B0202;

t4 polynucleotide kinase, NEB, M0201;

t4DNA ligase, NEB, M0202;

qiaquick gel recovery kit, Qiagen, 28704;

qiaquick PCR product recovery kit, Qiagen, 28104;

22.10 XCutsmart buffer, NEB, B7204;

23.10X NEBuffer 2.1,NEB,B7202；

t4DNA polymerase (3U/. mu.l), NEB, M0203;

25.AscI,NEB,R0558；

26.BbsI,NEB,R3559；

27.5X Q5 reaction buffer, NEB, M0491;

28.dNTPs 10mM,NEB,N0447；

q5DNA polymerase, NEB, M0491;

30.40% acrylamide: methylene bisacrylamide (29:1), Sangon, B546013;

31.10 XTBE buffer, Sangon, B540024;

32. urea, Sangon, a 510907;

33. ammonium persulfate, Sangon, a 00486;

TEMED (N, N' -tetramethylethylenediamine), Sangon, a 100761;

32.2 XUrea-TBE buffer, Sangon, C506046.

Example 1

(1) Prepare all joints

1. Preparation of the joint

A1-R-3' -biotin (SEQ ID NO.1), B1-F (SEQ ID NO.3), B2-F (SEQ ID NO.5), B3-F (SEQ ID NO.7), B4-F (SEQ ID NO.9) were phosphorylated using the following systems, respectively:

adding 3 μ l of reverse primers A1-F (SEQ ID NO.2), B1-R (SEQ ID NO.4), B2-R (SEQ ID NO.6), B3-R (SEQ ID NO.8), B4-R (SEQ ID NO.10) and 7ul of H2O, respectively, cooling to 25 ℃ at 0.1 ℃ per second on a PCR instrument for 2 minutes, and storing the adaptor in a refrigerator of minus 20.

(2) DNA end repair and 3' end tailing:

DNA end repair and 3' end addition of A. reaction System configuration was performed according to the following System

Incubate at 20 ℃ for 30min and 65 ℃ for 30 min.

(3) Ligation of the 3' -Biotin modified a1 linker and purification of the ligation product:

3. connection A1 Joint

Incubate at 20 ℃ for 4 hours DNA was purified using 80. mu.l Ampure XP beads, eluting at 100. mu. l H2O.

(4) Binding of DNA to a solid phase:

4. Immobilization of DNA to streptavidin magnetic beads:

50 μ l Dynabeads were taken^TMT1 magnetic beads to 1 eppendorf tube, adding 200. mu.l of 1 Xbinding and washing buffer, placing eppendorf tube on magnetic rack for 1min, discarding supernatant, washing repeatedly, resuspending Dynabeads with 100. mu.l of binding and washing buffer^TMT1 magnetic bead, adding 100. mu.l DNA, mixing, incubating at room temperature for 15min, placing eppendorf tube on magnetic frame for 1min, discarding supernatant, washing magnetic bead with 200. mu.l 1X binding and washing buffer for 2 times, washing magnetic bead with 100. mu.l 0.1M NaOH for 1 time

(5) Denaturation of double-stranded DNA into single strands

5. Denaturation of double-stranded DNA into single strands:

resuspend the beads with 100. mu.l 0.1M NaOH, incubate for 10 min at room temperature, place the eppendorf tube on the magnetic frame for 1-2 min, discard the supernatant, wash 1 time with 100. mu.L 0.1M NaOH, wash 2 times with 100. mu.l hybridization buffer.

(6) Hybridization of primers

6. Primer 1(SEQ ID NO.11) hybridizes:

mu.l of 10. mu.M primer 1 to 95. mu.l of hybridization buffer were diluted 5. mu.l, resuspended in magnetic beads, incubated at 85 ℃ for 30s, at 65 ℃ for 60s, cooled to 40 ℃ at a rate of 0.1 ℃/s, incubated at 40 ℃ for 5 minutes, Eppendorf tubes were placed on a magnetic frame, the supernatant was discarded, and the magnetic beads were washed 2 times with wash 1.

(7) Base extension and fixed length DNA extension:

7.23 cycles of reversible end reactions:

adding extension buffer solution, incubating at 65 ℃ for 1min, placing Eppendorf on a magnetic frame, discarding the supernatant, washing the magnetic beads with the washing buffer 1 for 2 times; adding 3' hydroxyl recovery buffer, incubating at 65 ℃ for 30s, placing the tube on a magnetic frame, discarding the supernatant, adding washing buffer 2, incubating at 65 ℃ for 30s, placing the Eppendorf tube on the magnetic frame, discarding the supernatant, washing the washing buffer 2 times, washing the washing buffer 1 time, and repeating the steps until 23 cycles are completed.

(8) Flattening the tail end:

flattening the 5' end suspension arm of DNA

The beads were washed 2 times with TE and the reagents resuspended beads were prepared as follows:

incubate at 30 ℃ for 30min, and TE wash the beads for 3 times.

DNA 5' end phosphorylation

The reagent resuspension beads were prepared as follows:

incubating at 37 ℃ for 30min, and washing the magnetic beads for 2 times with TE

(9) Selection of terminal sequences：

B1 Joint connection

The reagent reselection beads were prepared as follows:

incubating at 16 ℃ overnight, washing the beads with TE 2 times

PCR amplification

The PCR system was configured as follows:

the PCR tube was placed on a thermal cycler and the PCR reaction was performed according to the following procedure:

DNA agarose gel electrophoresis and DNA recovery

The DNA sequence generated in step 11 is shown in FIG. 2-A. A1.8% low melting point agarose TAE gel containing 50. mu.g/ml ethidium bromide was prepared, the PCR product was mixed with loading buffer, then added to the gel spot wells, run at 70V for 35 minutes, developed under 300nm UV light, as shown in FIG. 3-A, resulting in a 144bp band, which was cut and DNA purified using the gel extraction kit.

And (3) adding biotin to the end of the DNA5 for modification.

The PCR system was prepared as follows:

the PCR tube was placed on a thermal cycler and the following procedure was followed:

DNA recovery Using PCR clean-up kit, 40. mu.l water elution, concentration about 70 ng/. mu.l

14. First round of NGG PAM selection

200ng Biotin-DNA is fixed on streptavidin magnetic beads, and an enzyme digestion system is configured according to the following system: :

the Eppendorf tube was placed in a 37 ℃ water bath and incubated overnight.

B2 Joint connection

The reaction system was prepared as follows:

after 2 hours reaction at room temperature, the beads were washed 2 times with TE.

PCR amplification

The PCR reaction was prepared as follows:

the PCR tube was placed in a thermal cycler and the PCR reaction was performed according to the following procedure:

DNA agarose gel electrophoresis and DNA recovery

DNA generated in step 16A 1.8% low melting agarose TAE gel containing 50ug/ml ethidium bromide was prepared as shown in FIG. 2-B, the PCR product was mixed with loading buffer, added to the gel spot wells, run at 70V for 35 min, developed under 300nm UV light to obtain a 142bp band as shown in FIG. 3-B, the band was excised, and the DNA was purified using Qiagen gel recovery kit.

18. Second round NGG PAM selection:

the cleavage reaction was prepared as follows:

incubate at 37 degrees overnight.

DNA agarose gel electrophoresis and DNA recovery

2% low melting point agarose TAE gel containing 50ug/ml ethidium bromide was prepared, the digestion product was mixed with loading buffer, added to the gel spot wells, run at 35V for 90 minutes, developed under 300nm UV light, cut a band of about 81bp as shown in FIG. 2-C, DNA purified using Qiagen gel recovery kit, and eluted with 44.8. mu.l water.

Smoothing of hanging arms at DNA 5' ends

The reaction system was prepared as follows:

incubate at 30 ℃ for 30min, purify the DNA using 100. mu.l Ampure XP beads, and elute with 17.5. mu.l water.

Phosphorylation of DNA 5' end

The reaction system was prepared as follows:

incubate 30 minutes at 37 ℃ and 20min at 65 ℃.

(10) Removal of terminal sequences

22. The reaction system was prepared as follows:

incubate overnight at 16 ℃. 60ul Ampure XP beads purified DNA, 34.5. mu. l H2O eluted DNA.

23.PCR

The reaction system was prepared as follows:

the PCR tube was placed in a thermal cycler, and PCR reaction was performed according to the following procedure

DNA agarose gel electrophoresis and DNA recovery:

the DNA sequence generated in step 23 is shown in FIG. 2-C, and 1.8% low melting agarose TAE gel containing 50. mu.g/ml ethidium bromide is prepared, the PCR product is mixed with loading buffer, then added to the gel spot wells, run at 70V for 35 minutes, developed under 300nm UV light, the 143bp band is excised, and the DNA is purified using Qiagen gel recovery kit.

DNA 5' end with biotin modification.

The PCR system was prepared as follows:

DNA recovery was performed using Qiagen PCR product purification kit, eluted with 40. mu.l water at a concentration of about 70 ng/. mu.l

NGG PAM removal

the Eppendorf tube was placed in a 37 ℃ water bath and incubated overnight.

Steps 27 to 31 correspond to claims 1 to 7):

completion of DNA 3' end

The reaction system was prepared as follows:

incubate at 12 ℃ for 15min, and wash the beads 2 times with TE.

Phosphorylation of DNA 5' end

The reaction system was prepared as follows:

incubate at 37 ℃ for 30min, and TE wash the beads 2 times.

(11) Ligation of cloning adaptors

29. The reaction system was prepared as follows:

incubate at 16 ℃ overnight, and wash the beads 2 times with TE buffer.

PCR amplification:

the reaction system was prepared as follows:

DNA agarose gel electrophoresis and DNA recovery

The DNA sequence generated in step 30 is shown in FIG. 2-D. A1.8% low melting point agarose TAE gel containing 50. mu.g/ml ethidium bromide was prepared, the PCR product was mixed with loading buffer, then added to the gel spot wells, run at 70V for 35 minutes, developed under 300nm UV light to give a 141bp band below 200bp as shown in FIG. 2-D, the band was excised, and the DNA was purified using the Qiagen gel recovery kit.

(12) Short DNA Strand preparation

32. Short DNA Strand preparation

The reaction system was prepared as follows:

DNA was purified by 100% ethanol precipitation.

33. DNA purification by denaturing polyacrylamide gel electrophoresis

Preparing DNA modified polyacrylamide gel:

after coagulation, pre-run at 180V for 30min, mix DNA from step 32 with 2x urea-TBE buffer, denature at 90 ℃ for 5min, place directly on ice, start loading immediately, run at 180V for 40 hours, strip, stain with 0.1% toluene azure for 30min, decolour with water, excise more than 60nt portions, incubate at 65 ℃ overnight in TE.

(13) Construction of high throughput sequencing libraries

34. Constructing a high-throughput sequencing library, and analyzing the content of 20bp in the library;

A215 bp fragment was recovered by 1.8% DNA agarose gel electrophoresis.

The invention constructs the library of example 1and verifies 1) synthesis according to a template by example 1; 2) fixed length DNA; 3) and (4) selecting a terminal sequence.

Wherein figure 3: the results obtained in the terminal sequence selection and excision steps of the present invention were plotted by DNA agarose gel electrophoresis. A. After 23 times of 1bp extension and 3' hydroxyl recovery of the template DNA, a band of 144bp appears in the 5 th lane, and the sequence is shown in FIG. 2-A. B. After AscI digestion and ligation with B2 linker, a 142bp band appeared in lane 4, and its sequence is shown in FIG. 2-B. The DNA in Panel B was digested with AscI, resulting in a band of about 81bp in lane 5. D. The 4 th lane generated a 141bp band by BbsI cleavage to remove the terminal base, 5' end filling up and ligation of B1 adaptor, and the DNA sequence is shown in FIG. 2-D. D. After amplification with short primers and denaturing polyacrylamide gel electrophoresis, the DNA distribution in the third lane is about 59 bp-60 bp.

From FIG. 4, it can be seen that the 20bp sequence is aligned to the modified region of murine H3K4me3, and the resulting sequence from this method is derived from the starting material DNA of the present invention. As can be seen from FIG. 5A, C, the ratio of 20bp gRNA to starting DNA in the target region was substantially identical, demonstrating that the method of the present invention can construct a DNA library of a fixed length from the DNA in the target region.

From FIGS. 5-B, D, it can be seen that the gRNA of H3K4me3 region of mouse embryonic stem cells is followed by NGG sequence in about 96.65% of genome, which proves that the method can select DNA of specific terminal sequence from the library.

By combining the two points, the invention can construct a library of the mouse embryonic stem cell H3K4me3 region with fixed length and capable of being used together with spCas 9.

Example 2 preparation of a library of human H9 embryonic Stem cells

This example 2 constructs a fixed-length library using human embryonic stem cell H3K4me3 chromatin co-immunoprecipitation DNA, which was the same as in example 1 except that the template was different. The results are shown in FIGS. 6 and 7.

As shown in FIG. 6, the synthetic libraries, which are identical to the starting material, are located in the H3K4me3 region of human embryonic stem cells, and these DNAs are followed by NGG sequences, which demonstrates that the screening sequences of the present invention are also perfectly reproducible.

FIG. 6: for comparison of the case of synthesizing a 20bp sgRNA library of the H3K4me3 region of human embryonic stem cells from the template strand with the starting DNA: panel A is the case of starting material DNA alignment to human embryonic stem cells H3K4me3, and panel C is the case of the library alignment obtained to human embryonic stem cells H3K4me 3; panel B is the case where the starting material DNA follows the NGG sequence, and Panel D is the proportion of the resulting library that follows the NGG sequence; as can be seen in FIG. 6, the library is consistent with the starting DNA material in the proportion of the H3K4me3 modified region, and 20bp followed by the NGG sequence.

FIG. 7: the comparison of the synthetic library of the method of the invention and the synthetic library of the primer pool is shown in the figure: panel A is a proportion of DNA of each length obtained by synthesis, panel B is a proportion of DNA of 19 and 20bp followed by NGG sequence, panel C is a proportion of DNA of each length obtained by synthesis of a commercial primer pool, and panel D is a proportion of DNA of 20bp followed by NGG sequence obtained by synthesis of a commercial primer pool.

The current mainstream method for synthesizing a library by a gRNA is a primer pool synthesis method, and the inventor compares the method with the primer pool synthesis method, mainly shows whether a reference sequence is needed or not, whether a large-scale instrument is needed or not and whether the cost is low or not. The method can realize the synthesis of gRNAs without reference sequences which cannot be realized by a primer pool synthesis method, and simultaneously ensures that more gRNAs with functions can be realized by a final library. The invention does not need large-scale equipment, which provides convenience for the synthesis of gRNA in a common laboratory, and from the cost perspective, the invention manufactures a library containing about 13 ten thousand gRNAs, and through calculation, the cost for constructing the library is about 1000 yuan, which is far lower than the synthesis cost of a primer pool; from the attached figure 7, it can be seen that about 85% of the libraries of the present invention are 20bp and 19bp (both can be used as grnas because the sequences immediately following on the genome are NGG), about 89% of the grnas synthesized by the primer pool are 20bp, but more than 90% of 20bp and 19bp of the present invention are immediately following on the genome NGG sequences, and the proportion of the libraries synthesized by the primer pool is only 89%, so the method of the present invention has great potential to replace the current expensive primer pool synthesis method.

	This patent	Primer pool synthetic sequence
			Whether a reference genome is required	Does not need to use	Need to make sure that
Whether or not precision instruments are required	Does not need to use	Need to make sure that
			Cost actuarial	At present, 13 ten thousand pieces of the Chinese herbal medicine are synthesized, about 1000 yuan	1 ten thousand yuan, about 1 yuan and 1 piece

By combining example 1and example 2, it can be seen that the method of the present invention not only can robustly realize the synthesis of libraries of fixed length and specific terminal sequence according to the starting materials, but also can greatly save the cost.

Sequence listing

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Claims

1. A method for synthesizing a fixed-length and specific-end-sequence DNA library based on a template material, the method comprising:

1) primer hybridization: taking single-stranded DNA with a joint A1 fixed on a stationary phase with streptavidin, adding the single-stranded DNA into a DNA hybridization buffer solution and a primer 1, and carrying out gradient annealing;

the joint A1 comprises A1-R-3 '-biotin and A1-F, wherein A1-R-3' -biotin is a nucleotide sequence shown in SEQ ID NO.1, and A1-F is a nucleotide sequence shown in SEQ ID NO. 2;

the primer 1 has the sequence shown in SEQ ID NO: 11;

2) base elongation: then adding DNA polymerase and dNTPs with reversible chemical modification at the 3 'end into the product obtained in the step 1) to prolong the base, and then recovering the hydroxyl at the 3' end;

3) fixed length DNA extension: repeating the step 2), and circulating for 15-100 times;

4) flattening the tail end: removing the 5' -end by adding a single-stranded nuclease, wherein the single-stranded nuclease is mung bean acid nuclease, S1 nuclease or CEL I nuclease;

5) selection of terminal sequences: adding a DNA ligase and a linker B1, said linker B1 being a sequence comprising AscI, Acc65I, AccB1I, ApaI, Asp718I, AspS9I, AvaII, BamHI, BanI, BbeI, Bme18I, BmgT120I, BmiI, BshFI, BshNI, BsnI, Bsp120I, BspLI, BstEII, BstPI, BtsuI, BtsCI, Eco91I, EheI, FseI, HaeIII, KasI, KpnI, PhoI, SanDI, SfoI, SgsI, or SspDI cleavage sites to complete end selection; or

Replacing the base used for extension in the circulation corresponding to the position of the sequence to be selected, and then purifying the DNA with the complete length by polyacrylamide gel electrophoresis so as to complete the end selection;

6) removal of terminal sequences: adding a DNA ligase and a linker B3 to remove terminal sequences, wherein the linker B3 is a sequence comprising a Type II S restriction endonuclease site comprising BbsI, AcuI, AlwI, BaeI, BccI, BceAI, BbvI, BcoDI, BfuAI, BpuEI, BsaXI, BseRI, BsgI, BsmBI, BsmFI, BsmI, BspCI, BspMI, BspQI, BtgZI, EarI, Ecil, FauI, HgaI, HphI, HpyAV, MmeI, MnII, SapI, or StaStaStaNi;

7) ligation of cloning adaptor: adding DNA ligase and a linker B4 to connect a linker B4, wherein the linker B4 comprises B4-F and B4-R, wherein B4-F is a nucleotide sequence shown as SEQ ID NO.9, and B4-R is a nucleotide sequence shown as SEQ ID NO. 10;

wherein the single-stranded DNA having the linker A1 immobilized on the stationary phase having streptavidin in the step 1) is prepared by the following method:

1-1) acquisition of fragmented double-stranded DNA: reverse transcription of double-stranded DNA using sonicated or fragmented RNA to obtain fragments of 10bp to 40 Kbp;

1-2) DNA end repair and 3' end tailing, namely mixing the DNA obtained in the step 1-1) with DNA polymerase to carry out DNA 3' end flattening, and adding A or G to the 3' end;

1-3) ligation of 3' -Biotin modified A1 linker: mixing the product obtained in the step 1-2) with ligase, and adding a 3' -Biotin modified A1 linker; the ligase is blunt-end/TA DNA ligase;

1-4) purification of the ligation product: purifying double-stranded DNA by using 1X volume Ampure XP beads, or recovering DNA by using an ethanol or isopropanol precipitation method or a DNA silicon membrane column purification kit;

1-5) binding of DNA to a solid phase: mixing the DNA obtained in the step 1-4) with an isovolumetric stationary phase with streptavidin, and incubating at room temperature;

1-6) denaturation of double-stranded DNA into single strands: and (3) denaturing the DNA on the magnetic beads into single strands to obtain the DNA.

2. The method according to claim 1, wherein the cycle in the step 3) is 20 to 30 times.

3. The method according to claim 2, wherein the cycle in the step 3) is 20 to 23 times.

4. The method as claimed in claim 1, wherein the linker B1 in step 5) comprises B1-F and B1-R, wherein B1-F is the nucleotide sequence shown in SEQ ID NO.3 and B1-R is the nucleotide sequence shown in SEQ ID NO. 4.

5. The method as claimed in claim 1, wherein the linker B3 in step 6) comprises B3-F and B3-R, wherein B3-F is the nucleotide sequence shown in SEQ ID NO.7 and B3-R is the nucleotide sequence shown in SEQ ID NO. 8.

6. The method of claim 1, wherein the DNA hybridization buffer solution in step 1) is a5 XSSC buffer.

7. The method as claimed in claim 1, wherein the gradient annealing in step 1) is 85 ℃ for 30s,65 ℃ for 1min, and the temperature is slowly reduced to 40 ℃ at 0.1 ℃/s for 5min at 40 ℃.

8. The method of claim 1, wherein the step 2) further comprises:

the DNA polymerase is 9 ℃ N (D141A/E143A/A485L) DNA polymerase;

the dNTPs with reversible chemical modification are 3' -O-azidomethyl-dNTPs, 3' -O-allyl-dNTPs or 3' -O-allyloxycarbonylnes-dNTPs; or

The recovered 3' end hydroxyl is tri (2-carboxyacetic acid) phosphine hydrochloride or DTT.

9. The method of claim 1, wherein the step 2) further comprises adding an extension buffer while extending the base, incubating, washing the magnetic beads with washing buffer 1, then incubating with 3' hydroxyl-terminated recovery buffer, and washing with washing buffer 2.

10. The method according to claim 9, wherein in step 2),

the extension buffer composition includes 1. mu.M 3' -O-azidomethyl-dNTPs mix, 0.015. mu.g/ml 9 ℃ N DNA polymerase,50mM Tris pH 9.0,50mM NaCl,6mM MgSO4,1mM EDTA and 0.05% Tween 20;

the composition of the washing buffer 1 comprises 50mM Tris pH 9.0,50mM NaCl,1mM EDTA and 0.05% Tween 20;

the 3' hydroxyl recovery buffer composition includes 100mM TCEP,100mM Tris pH 9.0,100mM NaCl,50mM sodium ascorbate and 0.05% Tween 20; or

The composition of the wash buffer 2 included 100mM Tris pH 9.0,100mM NaCl and 0.05% Tween 20.

11. The method of claim 1, wherein the DNA ligase in step 5) is T4DNA ligase or blunt-ended/TA ligase, and the ligated B1 linker is used for terminal sequence selection.

12. The method of claim 1, wherein the step 5) further comprises: after the selection of the terminal sequence is carried out by using a B1 linker, the selection of the terminal sequence is carried out again by using a B2 linker, wherein the linker B2 comprises B2-F and B2-R, B2-F is the nucleotide sequence shown in SEQ ID NO.5, and B2-R is the nucleotide sequence shown in SEQ ID NO. 6.

13. The method of claim 1, wherein the DNA ligase in step 6) is T4DNA ligase or blunt-ended/TA ligase, and the ligated B3 linker is used for terminal excision.

14. The method of claim 1, wherein the DNA ligase in step 7) is T4DNA ligase or blunt-end/TA ligase, and ligated B4 is used for cloning adaptor ligation.

15. The method of claim 1, wherein the step 7) further comprises: for ligation of sequences required for molecular cloning, the fragments were cloned by existing cloning techniques into vectors for testing, the vector sequences being chimeric RNA sequences.

16. The method according to claim 1, wherein in the step 1-2), the DNA polymerase comprises: when the DNA is tailed at the 3' end with an A, the Klenow fragment (3' → 5' exo)^-) (ii) a When the 3' end of the DNA is added with a G tail, Taq DNA polymerase is used.

17. The method of claim 1, wherein the solid phase in step 1-5) comprises 2X B & W buffer resuspended T1 magnetic beads, Controlled porous Glass (Controlled Pore Glass), silica gel (silica gel), porous resin (Pore resin), microarray slides (Glass slides), silicon-based chips (silica chips), or nylon membrane (nylon membrane).

18. The method according to claim 1, wherein the denaturation in the step 1-6) is thermal denaturation or denaturation with a DNA denaturant.

19. The method of claim 18, wherein the DNA denaturant is sodium hydroxide, formamide, or urea.

20. The method of claim 1, wherein the fixed length is 15-100 bp.

21. The method of claim 20, wherein the fixed length is 20-30 bp.

22. The method of claim 21, wherein the fixed length is 20-23 bp.

23. The method of claim 1, wherein the specific end sequence is an NGG, NAG, or NAA sequence.

24. The method of claim 23, wherein the specific end sequence is SpCas 9:3' NGG; or SpCas9VRER mutant 3' NGCG; SpCas9EQR mutant 3' NGAG; SpCas9VQR mutant 3' NGAN or NGNG; 3' NNGRRT or NNGRR (N) of the SacAS9 mutant; AsCpf1and LbCpf1:5' TTTV; AsCpf1RR mutant 5' TYCV; LbCpf1RR mutant: 5' TYCV; AsCpf1RVR mutant 5' TATV; neisseria Meningitidis (NM):3' NNGATT; streptococcus Thermophilus (ST):3' NNAGAAW; or Treponema DentataA (TD):3' NAAAAC, of the desired terminal sequence of the Cas9 protein.