WO2018170614A1 - 基因组大片段直接克隆和dna多分子组装新技术 - Google Patents

基因组大片段直接克隆和dna多分子组装新技术 Download PDF

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WO2018170614A1
WO2018170614A1 PCT/CN2017/000483 CN2017000483W WO2018170614A1 WO 2018170614 A1 WO2018170614 A1 WO 2018170614A1 CN 2017000483 W CN2017000483 W CN 2017000483W WO 2018170614 A1 WO2018170614 A1 WO 2018170614A1
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exonuclease
nucleic acid
dna
acid molecule
rect
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WO2018170614A8 (zh
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张友明
王海龙
符军
阿德里安·弗朗西斯·斯图尔特
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Shandong University
Technische Universitaet Dresden
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Shandong University
Technische Universitaet Dresden
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Priority to KR1020197031095A priority patent/KR102488128B1/ko
Priority to CA3060585A priority patent/CA3060585A1/en
Priority to DK17901979.9T priority patent/DK3604524T3/da
Priority to EP17901979.9A priority patent/EP3604524B1/en
Priority to JP2020500937A priority patent/JP7106625B2/ja
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Definitions

  • the present invention relates to nucleic acid cloning and assembly methods, and more particularly to a novel method of deoxyribonucleic acid (DNA) cloning and assembly.
  • DNA deoxyribonucleic acid
  • DNA cloning is the core of molecular biology and biotechnology, and is the key technology for gene function research. As DNA sequencing technology continues to advance and sequencing costs continue to decrease, whole-genome sequencing becomes easier. It has been found that there are abundant untapped resources in the genome. Short DNA fragments can be easily obtained by PCR or chemical synthesis, but the traditional method of cloning DNA fragments larger than 10 kb depends on the construction and screening of DNA libraries. Traditional methods of library construction and screening are not only complicated, time-consuming, and laborious, but the target DNA fragments are often scattered on several different clones.
  • the above method requires a high amount of DNA fragments to be assembled, and it is necessary to use a target DNA fragment having a relatively high purity and concentration, and cannot be used for directly assembling a DNA fragment of interest from a mixture of enzymatic genomic DNA fragments.
  • RecET direct cloning A specific DNA region is cloned directly into the vector from genomic DNA, referred to herein as "direct cloning.”
  • the principle of RecET direct cloning technology is: Rac phage recombinant protein - full-length RecE and RecT can efficiently homologous recombination between linear DNA molecules in E. coli cells - line recombination.
  • RecE is a 5'-3' exonuclease
  • RecT is a single-stranded DNA annealing protein
  • protein-protein interaction between RecE and RecT is required for line recombination.
  • the line recombination efficiency of RecET combined is separate. 1000 times the effect.
  • the RecET direct cloning technology can directly capture genomic DNA fragments larger than 10 kb into an expression vector, and can also assemble 2 to 5 DNA fragments.
  • a suitable restriction enzyme site can be found in the genome to release the target DNA fragment, and then the restriction enzyme-cut genomic DNA and the linear vector are electrotransformed into E. coli cells expressing RecET recombinase.
  • the intracellular DNA fragment and the linear vector are homologously recombined by homologous arms at both ends to form a circular plasmid, and finally colonies containing the recombinant DNA molecule can be obtained by antibiotic screening and restriction analysis.
  • both the linear vector and the genomic DNA are prepared, it takes only 3 days to complete the direct cloning of the DNA fragment of interest.
  • RecET operates in the following 50kb DNA fragment was cloned from the bacterial genome 3-6,12 convenient, but it is difficult from the bacterial genomic clone larger DNA fragments, DNA fragments can not be cloned from the genome of mammalian . This is due to the fact that RecET direct cloning technology relies on the expression of RecET recombinase in cells. Only when the cloning vector and the target DNA fragment enter into an E.
  • genomic DNA fragments such as cloning of DNA fragments larger than 50 kb from the bacterial genome or cloning of genome fragments larger than 10 kb from mammals such as mice and humans. This is because the concentration of a certain segment of DNA in the genome is very low, which results in a small probability that the DNA fragment of interest and the cloning vector enter a cell at the same time of transformation.
  • mice due to the influence of physical shearing force during the preparation of genomic DNA, the larger the target DNA fragment to be cloned, the lower the concentration in the genome, and finally the probability of the target DNA fragment and the cloning vector entering a cell together.
  • the genomes of mice (2800Mb) and humans (3200Mb) are nearly three orders of magnitude larger than the bacterial genome (5Mb), so genomic DNA fragments of the same size are much lower in mammalian genome than bacteria, so mammals are being performed.
  • the probability that the target DNA fragment enters an E. coli cell simultaneously with the cloning vector is also much lower.
  • ExoCET technology not only clones >100 kb DNA fragments directly from the bacterial genome, but also clones >50 kb DNA fragments from mammalian cells and human blood. ExoCET technology also efficiently assembles at least 20 DNA fragments to form a complete plasmid.
  • ExoCET Like the recET direct cloning technology, ExoCET has the advantage of being superior in fidelity, does not destroy the haplotype of DNA, and the target DNA is directly cloned into a plasmid vector to facilitate expression studies. In addition, ExoCET is more efficient than Gibson assembly because Gibson relies on circular DNA molecules produced by in vitro assembly. Moreover, the Gibson assembly in the direct cloning will produce a very serious background caused by the emptying of the empty vector itself, which may be the reason why Jiang et al. 11 performed PCR pre-screening before identifying the recombinant DNA; also Zhou et al. 13 When cloning from the Streptomyces conglobatus genome by Gibson assembly to pass the conglobatin gene cluster, it is necessary to remove the genomic DNA fragment of 20 kb or less produced by restriction enzyme digestion by agarose gel electrophoresis.
  • ExoCET can also be used to directly clone DNA fragments from mammalian genomes including blood, disease-associated cell lines, etc. to facilitate haplotype studies of SNPs and to rapidly construct haplotypes for nuclease-mediated human stem cell targeting.
  • Gene (HIT) targeting vector The importance of human stem cells isolated from patients, cord blood or somatic cell reprogramming in biomedical research has received increasing attention, and research on the precise modification of stem cell genome has received widespread attention. Transforming the human genome is more challenging than structuring the genome of experimental mice because human genetic diversity is complex. Syngeneic (sequence similarity) the importance of homologous recombination many years ago people mouse embryonic stem cells in gene targeting when he realized 14.
  • ExoCET is an effective way to quickly obtain ideal homology arms. Unlike the method of amplifying homology arms from the genome by PCR, ExoCET is not limited by fragment size, does not introduce mutations, and is capable of maintaining a haplotype of DNA.
  • the ends of the homology arms can also be selected according to the manner of genotyping (such as Southern hybridization or ligation PCR), so the length of the homology arms can be optimized.
  • ExoCET therefore offers advantages for individualized genomic surgery, especially when combined with CRISPR/Cas9 15 .
  • ExoCET can also be used as the most reliable method for genotyping a modified genome, while Southern hybridization and ligation PCR produce false positive signals.
  • ExoCET's ability to selectively capture large fragments of DNA from complex genomes has become a means of disease diagnosis and pathology testing, such as capturing DNA sequences directly from individualized medicine or isolating DNA viruses from patient samples.
  • ExoCET will be widely used in functional genomics and comparative genomics research, especially for direct cloning of prokaryotic synthesis pathways or assembly of multiple DNA molecules for synthetic biology.
  • the invention provides a method of homologous recombination, the method comprising treating two or more target nucleic acid molecules with a first exonuclease, and then subjecting the treated target nucleic acid molecule to a second nucleic acid Homologous recombination is carried out in the presence of an exonuclease and an annealing protein, wherein at least one sequence homologous region is shared between target nucleic acid molecules in which homologous recombination occurs.
  • the invention provides a method of homologous recombination, wherein the method comprises treating a first nucleic acid molecule and a second nucleic acid molecule with a first exonuclease, and then treating the treated first nucleic acid molecule with Two nucleic acid molecules in the second exonuclease and annealing protein Homologous recombination is carried out in the presence of at least one sequence homologous region between the first nucleic acid molecule and the second nucleic acid molecule.
  • the invention provides a method of assembling a linear nucleic acid molecule, the method comprising treating two or more nucleic acid molecules with a first exonuclease, and then subjecting the treated nucleic acid molecule mixture to a second Homologous recombination is carried out in the presence of an exonuclease and an annealing protein, wherein each nucleic acid molecule shares at least one sequence homologous region with a nucleic acid molecule that forms a contig in the resulting assembled product.
  • the invention provides a method of cloning genomic DNA, wherein the method comprises treating a genomic DNA fragment mixture and a linear cloning vector using a first exonuclease, and then subjecting the target DNA fragment in the mixture of the treated genomic DNA fragments And the linear cloning vector performs homologous recombination in the presence of a second exonuclease and an annealing protein, wherein at least one sequence homologous region is shared between the target DNA fragment and the linear cloning vector in the genomic DNA fragment mixture.
  • sequence homology region may be at the end or end of the target nucleic acid molecule, preferably at least one homologous region at the end of the target nucleic acid molecule, more preferably the homologous region is at the end of the target nucleic acid molecule.
  • the homologous region has a length of at least 6, at least 10, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80 Nucleotides, preferably 25, 40 or 80, most preferably 80 nucleotides.
  • the first exonuclease may be a 5' to 3' exonuclease or a 3' to 5' exonuclease, preferably a T4 DNA polymerase, a DNA polymerase
  • T4 DNA polymerase a DNA polymerase
  • the Klenow fragment of I, the T5 exonuclease, and the T7 exonuclease are most preferably T4 DNA polymerase or T5 exonuclease.
  • the first exonuclease treatment comprises an in vitro ligation step of causing two or more target nucleic acid molecules or the first nucleic acid molecule and the The two nucleic acid molecules are linked together or the target DNA fragment in the mixture of the treated linear cloning vector and the genomic DNA fragment is ligated.
  • the first exonuclease treatment comprises a digestion and annealing step, wherein the enzymatic cleavage of the different nucleic acid molecules can be performed separately or simultaneously, such as a mixture in a single sample. in.
  • the first exonuclease treatment further comprises A step of adding DNA polymerase, dNTP and DNA ligase.
  • the first exonuclease treatment further comprises adding a DNA polymerase having 3'-5' exonuclease activity.
  • the single-stranded annealing protein comprises RecA, RAD51, Red ⁇ , RecT, Plu ⁇ or RAD52, preferably wherein the annealing protein is RecT, more preferably, the RecT is recombinantly expressed.
  • the annealing protein is RecT, preferably, the RecT is recombinantly expressed.
  • the host cell may be a yeast cell, preferably the yeast cell is a Saccharomyces cerevisiae cell; or a bacterial cell, preferably the bacterial cell is Bacillus subtilis or Escherichia coli.
  • said host cell expresses an exonuclease, preferably a second exonuclease, and an annealing protein.
  • said host cell expresses an exonuclease, an annealing protein and Red gamma, preferably said host cell further expresses RecA, most preferably said host cell expresses RecE, RecT, Red ⁇ and RecA.
  • the host cell is an E. coli cell expressing full-length RecE and RecT
  • the host cell is an E. coli cell expressing full-length RecE, RecT and Red ⁇
  • the host cell is an E. coli cell expressing full-length RecE, RecT, Red gamma, and RecA.
  • said host cell is an E. coli cell expressing truncated RecE and RecT.
  • said host cell is an E. coli cell expressing Red alpha and Red beta.
  • the host cell expresses an exonuclease, preferably a second exonuclease, an annealing protein, Red gamma and/or RecA, on the plasmid vector and/or chromosome, preferably, Expression is carried out by a plasmid vector, most preferably by a plasmid vector and a chromosome.
  • the target nucleic acid molecule or the target DNA fragment is linear, preferably selected from a DNA fragment cleaved with an endonuclease, a PCR amplified DNA fragment, a genomic DNA fragment, a cDNA library member Fragments derived from BAC and cloning vector fragments.
  • the endonuclease is a restriction endonuclease or a programmable endonuclease, such as Cas9.
  • the target nucleic acid molecule comprises a length of 0.5 kb or longer (eg, 1 kb or longer, 2.5 kb or longer, 4 kb or longer, 5 kb or longer, 7.5 kb or Sequences longer, 10 kb or longer, 15 kb or longer, 20 kb or longer, 25 kb or longer, 40 kb or longer, 50 kb or longer, 75 kb or longer or 100 kb or longer).
  • 0.5 kb or longer eg, 1 kb or longer, 2.5 kb or longer, 4 kb or longer, 5 kb or longer, 7.5 kb or Sequences longer, 10 kb or longer, 15 kb or longer, 20 kb or longer, 25 kb or longer, 40 kb or longer, 50 kb or longer, 75 kb or longer or 100 kb or longer.
  • the two or more target nucleic acid molecules, the first target nucleic acid molecule and the second target nucleic acid molecule or target DNA fragment comprise one or more PCR amplified DNA fragments, genomic DNA fragments, cDNA library members, and / or a fragment derived from BAC;
  • the first exonuclease has a 3'-5' exonuclease activity, preferably a T4 DNA polymerase;
  • exonuclease treatment is carried out in vitro in the absence of dNTPs
  • the second exonuclease is full length RecE;
  • the annealing protein is RecT
  • said homologous recombination is carried out in a bacterial host cell expressing said full length RecE and RecT, preferably E. coli.
  • the two or more target nucleic acid molecules comprise one or more PCR amplified DNA fragments, genomic DNA fragments, cDNA library members, and/or derived from BAC a fragment, a linear plasmid and/or a cloning vector fragment, preferably three or more linear plasmids and/or cloning vector fragments;
  • the first exonuclease comprises a Gibson assembly
  • the second exonuclease is full length RecE;
  • the annealing protein is RecT
  • said homologous recombination is carried out in a bacterial host cell expressing said full length RecE and RecT, preferably E. coli.
  • the two or more target nucleic acid molecules comprise three or more PCR amplified DNA fragments, genomic DNA fragments, cDNA library members, or fragments derived from BAC, linear plasmids and/or cloning vectors, preferably three Or more linear plasmids and/or cloning vector fragments;
  • said first exonuclease treatment comprises Gibson assembly
  • the second exonuclease is full length RecE;
  • the annealing protein is RecT
  • said homologous recombination is carried out in a bacterial host cell expressing said full length RecE and RecT, preferably E. coli.
  • kits comprising the first exonuclease and the second exonuclease described in the method of any of the preceding or the first exonuclease and the second nucleic acid in the method of any of the foregoing Dicer nucleic acid.
  • kits comprising the first exonuclease and the second exonuclease or the nucleic acid encoding the first exonuclease and the second exonuclease as described in the method of any of the preceding claims, preferably
  • the kit further comprises a host cell expressing a second exonuclease, preferably the host cell expresses an exonuclease, an annealing protein and Red gamma, preferably the host cell further expresses RecA, most preferably The host cell expresses RecE, RecT, Red ⁇ and RecA, the host cell may be a yeast cell, preferably the yeast cell is a Saccharomyces cerevisiae cell; or a bacterial cell, preferably the bacterial cell is Bacillus subtilis or Escherichia coli, the host The cells are expressed by a plasmid vector, preferably by a plasmid vector and, preferably, by a plasmid vector and a chromosome
  • said first exonuclease is 3'-5' Exonuclease activity DNA polymerase, such as T4 DNA polymerase, Klenow fragment of DNA polymerase I, T5 exonuclease or T7 exonuclease, the second exonuclease is full length RecE.
  • DNA polymerase such as T4 DNA polymerase, Klenow fragment of DNA polymerase I, T5 exonuclease or T7 exonuclease
  • the second exonuclease is full length RecE.
  • kit according to any one of the preceding claims, wherein the kit further comprises a host cell expressing a second exonuclease, preferably comprising a nucleic acid encoding full length RecE, RecT, Red gamma and RecA.
  • kit according to any of the preceding claims, wherein said kit further comprises one or more pre-prepared linear carriers.
  • Figure 1 Combined use of in vitro assembly and RecE/RecT promotes the efficiency of direct cloning.
  • ExoCET increases the efficiency of direct cloning.
  • GB05-dir (ExoCET) containing pSC101-BAD-ETgA-tet was used, or T4pol treatment (ETgA) was not used, or arabinose induction (T4pol) was not used.
  • Figure 2 Effect of different exonucleases on direct cloning of the lux gene cluster.
  • the p15A-cm vector and the B. luminifera genomic DNA were treated with exonuclease, annealed and then electrotransformed into arabinose-induced E. coli GB05-dir.
  • (a) Initial detection of different exonucleases.
  • (bd) Optimize the concentrations of Kle, T5exo and T7exo.
  • e Comparison of cloning efficiency after 20 min digestion with T4pol, Kle, T5exo and T7exo at optimal concentrations.
  • (f) Optimize the temperature and time of T4pol digestion (0.02 U ⁇ l -1 ). Error line, sd; n 3.
  • p15A-cm vector and B. luminifera genomic DNA were digested with 0.02 U ⁇ l -1 T4pol in different ways (A, B, C) was post-annealed to arabinose-induced E. coli GB05-dir.
  • Figure 4 Flowchart of preparation of a linear cloning vector and ExoCET direct cloning technique.
  • a p15A-cm vector was prepared by PCR amplification using a primer of 80 nucleotides.
  • FIG. 5 Position of the 80 bp homology arm between the p15A-cm and 14 kb lux genomic DNA fragments: (A) both homologous arms are at the extreme; (B, C) one located 1 kb from the end The other is at the very end; (D) is located 1 kb from the end. The reaction conditions are the same as in Figure 1f. (b) The number of colonies obtained by ETgA, T4pol or ExoCET using the above four homology arms.
  • FIG. 6 Direct cloning of a 106 kb salinomycin gene cluster using Streptomyces cerevisiae S. albus genomic DNA digested with EcoRV or Cas9.
  • the salinomycin gene cluster was cloned from the EcoRV or Cas9gRNA2/Cas9-gRNA7 digested genomic DNA into the pBeloBAC11 vector using ExoCET.
  • the homology arm (blue) was first inserted into the BAC vector and the BAC vector was linearized by BamHI digestion as a direct cloning vector. The length of the homology arm is indicated at the end of the genomic DNA fragment.
  • PvuII restriction analysis of recombinant DNA the correct clones are indicated by arrows.
  • FIG. 7 Comparison of the efficiency of ExoCET and Gibson assembly.
  • (c) Schematic diagram of assembling a plurality of DNA fragments into a plasmid by T4pol, ExoCET, Gibson, and Gibson+ETgA.
  • the DNA fragment has a size of 1.0 kb to 5.4 kb
  • the assembled p5A plasmid has a size of 29.8 kb to 54.9 kb and has chloramphenicol resistance (cm).
  • (d) The number of clones obtained by the multi-fragment assembly experiment and the correct rate. The in vitro assembled product was transformed into arabinose induction (ExoCET or Gibson + ETgA) or not induced (T4pol or Gibson) in GB05-dir containing pSC101-BAD-ETgA-tet.
  • FIG. 8 Construction of HIT (haplotype isogenic targeting) vectors for DPY30 using mammalian genomic DNA.
  • FIG. 9 Construction of HIT (haplotype isogenic targeting) vectors for Dpy30 using mouse cell genomic DNA.
  • Figure 10 shows the genotyping of mammalian cells using ExoCET.
  • DNA recombination engineering is a genetic engineering technique for modifying DNA molecules in E. coli cells using 18-22- mediated homologous recombination of phage syn/exo proteins (mainly Red ⁇ and Red ⁇ ).
  • Recombinant DNA engineering techniques are found in the beginning E.colisbcA (recBC inhibitor) strain, the strain has 23 active homologous recombination between DNA molecules with high homology arm mediated (homology boxes) a.
  • the sbcA strain was discovered in a classic assay by AJ Clark looking for a homologous recombination pathway in E. coli. He used the recBC strain, which is very sensitive to DNA damage, to screen its inhibitors and found sbcA mutant strains 24-26 with RecE and RecT expression activities.
  • RecE and RecT are expressed by Rac phage integrated on the chromosome, which function identically to phage Red ⁇ and Red ⁇ 27 and only 280 amino acids at the C-terminus of RecE protein 28-30 are expressed in the sbcA mutant strain.
  • Truncated RecE and Red ⁇ (266 amino acids) is similar to a 5'-3 'exonuclease 31, RecT and Red ⁇
  • SSAP single strand annealing protein
  • RecE/RecT and Red ⁇ /Red ⁇ belong to the 5'-3' exonuclease/SSAP syn/exo protein pair 21,33 , and the homologous recombination of the double-stranded DNA of the protein-protein interaction potential specific to each pair of proteins Required 29, 34, 35 .
  • Red ⁇ /Red ⁇ -mediated homologous recombination occurs primarily on the replication fork and requires replication to proceed 36,37 .
  • the inventors initially discovered recombinant engineering techniques through truncated RecE/RecT, the inventors later used Red ⁇ /Red ⁇ to modify DNA molecules because the latter were more efficient, 16, 38-42 .
  • RecE / RecT Characteristics RecE / RecT, and found that RecE N terminal 600 amino acid residues to a recombinant replication-dependent activity becomes dependent replication of 3. Therefore, two linear DNA molecules can form a circular plasmid by efficient homologous recombination through a short homologous arm.
  • wire lines having different recombination mechanisms range of applications, such as direct cloning from genomic DNA or a large fragment 3-6,12 multiple DNA fragments 15, 43 of the assembly.
  • the present invention provides a method for homologous recombination (line recombination) between two or more target linear nucleic acid molecules sharing at least one sequence homology region, wherein the method comprises treating a mixture of target linear nucleic acid molecules using a first exonuclease; and then subjecting the treated target linear nucleic acid molecule to homologous recombination in the presence of a second exonuclease and an annealing protein.
  • the second exonuclease may be RecE, and the amino acid sequence of full length RecE from E. coli K12 is disclosed in WO2011/154927.
  • the second exonuclease may also be a truncated RecE comprising RecE protein 29 consisting of amino acids 588-866, 595-866, 602-866 or 606-866.
  • the annealing protein used in the methods of the invention is the annealed protein disclosed in WO2011/154927.
  • the annealing protein is RecT or a fragment thereof (derived from Rac phage). More preferably, the annealing protein is full length RecT and the second exonuclease is full length RecE.
  • any other suitable annealing protein can be used as long as the annealing protein interacts with the exonuclease used. Examples of other suitable phage annealing proteins are provided in WO 02/062988.
  • Line recombination can occur in certain host cells lacking RecT expression, such as E. coli strain GB2005, possibly due to the presence of certain endogenous RecT-like activities.
  • the efficiency of line recombination mediated by full length RecE was significantly increased in the presence of RecT.
  • the methods of the invention may be affected in whole or in part in a host cell.
  • Suitable host cells include cells of many species, including parasites, prokaryotes, and eukaryotes, but bacteria such as Gram-negative bacteria are preferred hosts. More preferably, the host cell is an enteric bacterial cell such as Salmonella, Klebsiella, Bacillus, Neisseria, Photorhabdus or Escherichia. Coli) cells (the method of the invention plays an effective role in all E. coli strains that have been tested).
  • a preferred host cell is E. coli K12. It should be noted, however, that the methods of the invention are equally applicable to eukaryotic cells or organisms, such as fungal, yeast, plant or animal cells. This system has been shown to be functional in mouse ES cells and it is reasonable to speculate that it is also functional in other eukaryotic cells.
  • the host cell is an isolated host cell, but unisolated host cells can likewise be used.
  • the host cell of the present invention comprises a nucleic acid encoding an exonuclease (preferably full length RecE), an annealing protein (preferably RecT) and Red gamma.
  • the host cell further comprises a nucleic acid encoding RecA.
  • the host cell expresses RecE, RecT and Red gamma, and optionally RecA.
  • the host cell expresses RecE, RecT, Red ⁇ and RecA.
  • the exonuclease, annealing protein, Red gamma and/or RecA of the present invention may be recombinantly expressed from a foreign cell in a host cell, for example, by a vector transformed into a host cell.
  • a suitable vector is the pSC101 plasmid, although other suitable vectors can also be used.
  • Any suitable promoter can be used to drive expression of these proteins.
  • an inducible promoter such as an arabinose-inducible promoter (P BAD ) or a rhamnose-inducible promoter (P RhaSR ) is preferred . These promoters are well known in the art.
  • the host cell of the present invention expresses an exonuclease, an annealing protein, Red gamma and/or RecA by an inducible promoter on a plasmid vector or a chromosome.
  • the exonuclease, annealing protein, Red gamma and/or RecA are expressed in the host cell by a plasmid vector.
  • the exonuclease, annealing protein, Red gamma and/or RecA are simultaneously expressed in the host cell by the plasmid vector and the chromosome.
  • the genome of the E. coli K12 host cell includes an endogenous copy of the full-length recE gene and the recT gene, which are present in the Rac phage that has been integrated into the host genome.
  • the gene since the gene is silent, the expression of full-length RecE cannot naturally occur from the integrated gene.
  • the method can be carried out in the absence of an endogenous recE gene.
  • Host cells transformed with a nucleic acid molecule encoding an exonuclease as described above are also provided.
  • the exonuclease is expressed by the nucleic acid molecule, and thus the present invention also provides a host cell expressing the exonuclease enumerated in the method of the present invention.
  • the exonuclease is preferably expressed under the control of an inducible promoter, such as an arabinose-inducible promoter (P BAD ) or a rhamnose-inducible promoter (P RhaSR ).
  • an inducible promoter such as an arabinose-inducible promoter (P BAD ) or a rhamnose-inducible promoter (P RhaSR ).
  • the methods of the invention are affected in whole or in part in vitro.
  • purified 5' to 3' exonuclease and annealing protein preferably purified RecE and RecT proteins
  • E. coli cells expressing the 5' to 3' exonuclease and annealing protein can be used. Extract.
  • Linear recombination requires that at least one sequence homology region must be shared between the target linear nucleic acid molecules through which homologous recombination occurs.
  • the first target nucleic acid molecule shares a sequence homology region with the second target nucleic acid molecule to recombine the line between the first and second target nucleic acid molecules to produce a linear product.
  • each linear nucleic acid shares a linear nucleic acid that forms its neighbor in the linear product of the in-line recombination reaction. Sequence homology region.
  • the cyclic product of each linear nucleic acid recombined with the on-line Linear nucleic acids that form their neighbors share a sequence homologous region.
  • the first target nucleic acid molecule and the second target nucleic acid molecule share two sequence homology regions to recombine a line between the first and second target nucleic acid molecules to form a circular molecule.
  • At least one homology arm is at the extreme end of each linear segment.
  • these sequence homologous regions or "homologous arms” produce the optimal configuration, thus constructing these homology arms for recombination ring. Line recombination can occur when the homology arm is not at the end, but the efficiency is reduced.
  • at least one region of the homolog is located at the outermost end of one or both ends of the target nucleic acid molecule. In some embodiments, the homologous region is internal to the certain target nucleic acid molecule.
  • the sequence homology region of the present invention has a length of at least 4, at least 6, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 nuclei. Glycosylate.
  • the sequence homology regions are 4-6, 6-9, 6-30, 6-100, 10-20, 20-29, 20-40, 20-50, 10-100, 25 -30, 25-40, 25-50, 30-40, 30-50, 40-50, 40-80 or more than 80 nucleotides.
  • the efficiency of homologous recombination usually increases with the length of the homology arms used, so longer homology arms can be used.
  • “Homologous" between two nucleic acid molecules means that when the sequences of two nucleic acid molecules are aligned, there are many nucleotide residues that are identical at the same position in the sequence. The degree of homology is easily calculated (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing.
  • the methods of the invention comprise joining together a plurality of linear nucleic acid molecules to form a circular nucleic acid molecule, such as a circular plasmid.
  • a circular nucleic acid molecule such as a circular plasmid.
  • Each of the target nucleic acid molecules shares a homologous region with a target nucleic acid molecule that forms a proximity in the produced cyclic product, and is subjected to line recombination in accordance with the method of the present invention.
  • the number of target nucleic acid molecules is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.
  • At least one of the target linear nucleic acid molecules comprises a selection marker to allow selection of the correct recombinant.
  • a selection marker can be used in the present invention.
  • the selection marker is an antibiotic resistance gene, eg, a resistance gene for chloramphenicol, ampicillin, kanamycin, or blasticidin.
  • the target linear nucleic acid molecule can be derived from any suitable source.
  • nucleic acid sequences from eukaryotes or prokaryotes can be included.
  • the first target linear nucleic acid molecule is genomic DNA.
  • the genomic DNA is a fragment of genomic DNA.
  • the genomic DNA preferably includes a sequence of interest.
  • a genomic DNA fragment can be obtained by cleavage or digestion of genomic DNA (eg, using a restriction enzyme) to comprise the entire sequence of interest.
  • the first target linear nucleic acid molecule (eg, a genomic DNA fragment, a cDNA library member, or a BAC-derived fragment) comprises 2 kb or longer (eg, 2.5 kb or longer, 4 kb or longer, 5 kb or longer, 7.5 kb or longer, 10 kb or longer, 15 kb or longer, 20 kb or longer, 25 kb or longer, 40 kb or longer, 50 kb or longer, 75 kb or longer or 100 kb or longer ) the sequence of interest.
  • the sequence of interest is the entire region between the homology arms at either end of the first target linear nucleic acid molecule.
  • the methods of the invention can be used to clone DNA regions directly from the human or non-human animal genome, for example, for health research or for regenerative treatment modified by gene targeting.
  • the first target nucleic acid molecule comprises or consists of a genomic DNA fragment from a human or non-human animal.
  • the genomic DNA fragment can include a sequence of interest, such as a gene comprising a mutation, wherein the mutation results in a disease or condition and the modification of the mutation to a wild type sequence can treat or prevent the disease or condition.
  • the second target nucleic acid molecule is preferably a linearized cloning vector.
  • the method comprises generating a first target nucleic acid molecule by digesting or shearing genomic DNA to obtain a linear genomic DNA fragment comprising the sequence of interest, followed by The first exonuclease treats a mixture of the genomic DNA fragment and the linear cloning vector, and the treatment comprises the steps of cleavage of the target nucleic acid molecule and annealing to ligate the target nucleic acid molecule after digestion, and then co-electrically converting the mixture of the treated nucleic acid molecules Into the host cell.
  • the second target nucleic acid molecule preferably comprises a selection marker.
  • the methods of the disclosure comprise the step of ligation of DNA molecules in vitro.
  • the in vitro ligation step comprises exonuclease digestion followed by annealing.
  • the exonuclease is T4 polymerase.
  • the in vitro ligation step comprises Gibson assembly.
  • the in vitro ligation step comprises DNA synthesis by a DNA polymerase with or without exonuclease followed by annealing.
  • the in vitro ligation step comprises an annealing promoted by a single strand annealing protein such as RecA/RAD51, Red[beta], RecT, Plu[beta] or RAD52.
  • a single strand annealing protein such as RecA/RAD51, Red[beta], RecT, Plu[beta] or RAD52.
  • the host cell for homologous recombination is an E. coli cell.
  • the host cell for homologous recombination is an E. coli cell expressing full length RecE and/or ReeT.
  • the host cell for homologous recombination is an E. coli cell expressing full length RecE, RecT and/or Red gamma.
  • the host cell for homologous recombination is an E. coli cell expressing truncated RecE, RecT and/or Red gamma.
  • the host cell for homologous recombination is any bacterial host cell that expresses full length RecE and/or RecT.
  • the host cell for homologous recombination is an E. coli cell expressing Red alpha, Red beta and/or Red gamma.
  • the host cell for homologous recombination is a Saccharomyces cerevisiae cell.
  • kits for use in the present invention comprise a nucleic acid encoding an exonuclease as described herein.
  • the kit comprises an exonuclease as described herein.
  • the first exonuclease is T4 DNA polymerase (T4pol), Klenow fragment of DNA polymerase I (Kle), T7 DNA polymerase (T7 DNA polymerase; T7pol) Exonuclease III (ExoIII), Phusion DNA polymerase (Phu), T5 exonuclease (T5exo), T7 exonuclease (T7exo) and Lambda An exonuclease (lambda exonuclease; ⁇ exo), the second exonuclease is full-length RecE.
  • T4pol T4 DNA polymerase
  • Klenow fragment of DNA polymerase I Kle
  • T7 DNA polymerase T7 DNA polymerase
  • T7pol Exonuclease III
  • the kit comprises a host cell as described herein.
  • a host cell in a kit comprises a nucleic acid encoding a full length RecE, RecT, Red gamma, and RecA described herein under the control of an inducible promoter.
  • the kit may also include one or more pre-prepared linear cloning vectors.
  • the first and second target nucleic acid molecules are linear, and the method further comprises, in the presence of a 5' to 3' exonuclease and an annealing protein, the first sum
  • the second target nucleic acid molecule is contacted with one or more other linear target nucleic acid molecules (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, at least 10, at least 20 other target nucleic acid molecules) to produce Linear or cyclic product.
  • homologous recombination between the first and second target nucleic acid molecules and one or more other target nucleic acid molecules results in the production of a gene, operon, chromosome or whole genome.
  • Synthetic biological assembly of DNA nucleic acids has been used to generate genes, operons, chromosomes, or recently used to generate entire genomes.
  • the combination of the first exonuclease and the second exonuclease significantly increases the assembly efficiency of the linear nucleic acid molecule, and the present invention becomes a kind of synthetic biological DNA assembly in commercial and research. Preferred method.
  • haplotype isogenic targeting vector which can directly clone a 5 to 10 kb DNA fragment from a mammalian genome using the method of the present invention as an isogenic homology arm, and these DNA fragments are not only the same gene. Moreover, the polymorphic haplotype is maintained, so the inventor calls it a haplotype isogenic targeting vector, namely "HIT" (haplotypic Isogenic targeting) vector. The screening marker and other functional elements are then inserted into the HIT vector by recombinant engineering to obtain a vector for targeting.
  • HIT haplotypic Isogenic targeting
  • Another preferred application of the invention is the genotyping of mammalian cells.
  • the DNA fragment containing the complete targeting element is cloned from the genome of the possible target embryonic stem cells by the method of the present invention, and the recombinant plasmid obtained by the cloning is subjected to restriction analysis and DNA sequencing, and the cell is successfully determined according to the result.
  • GB05-dir is integrated with the P BAD -ETgA operon on the chromosome of GB2005 (recE, recT under the control of P BAD promoter) Red ⁇ and recA) 3 .
  • GB08-red integrates the P BAD -gbaA operon on the chromosome of GB2005 (red ⁇ , red ⁇ , red ⁇ and recA under the control of P BAD promoter) 16 .
  • pSC101-BAD-ETgA-tet 3 band A temperature-sensitive plasmid with a tetracycline resistance gene and the P BAD -ETgA operon, which can replicate at 30 ° C, but does not replicate at 37 ° C, so it can be easily removed by changing the temperature without selection pressure.
  • the Gram-negative bacteria Photobacterium phosphoreum ANT-2200 and Photorhabdus luminescens DSM15139 were cultured overnight in 50 ml of medium.
  • the cells collected by centrifugation were suspended in 8 ml of 10 mM Tris-HCl (pH 8.0), then 500 ul of 20 mg ml -1 proteinase K and 1 ml of 10% SDS were added, and the bath was heated at 50 degrees for 2 hours until the solution became clear.
  • Genomic DNA was obtained by phenol chloroform extraction and ethanol precipitation, and finally dissolved in 10 mM Tris-HCl (pH 8.0).
  • the Gram-positive bacterium Streptomyces albus DSM41398 was cultured at 30 degrees in 50 ml TSB medium for 2 days.
  • the genomic DNA was extracted according to the method of Reference 45. The main procedure was as follows: the bacteria were suspended in 8 ml of SET (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 8.0), 10 mg of lysozyme was added, and the bath was heated for 1 hour at 37 degrees, and then 500 ul of 20 mg ml was added . 1 Proteinase K and 1 ml of 10% SDS, warmed at 50 degrees for 2 h until the solution became clear, then 3.5 ml of 5 M NaCl was added. Genomic DNA was obtained by phenol chloroform extraction and ethanol precipitation, and finally dissolved in 10 mM Tris-HCl (pH 8.0).
  • Genomic use of mouse melanoma cells B16, human embryonic kidney cells 293T and human blood The Qiagen Blood&Cell Culture DNA kit was extracted and the steps were slightly modified.
  • the lysed cells were treated with proteinase K, extracted with phenol chloroform and ethanol precipitated to obtain genomic DNA, and finally dissolved in 10 mM Tris-HCl (pH 8.0).
  • the genomic DNA of Bacillus luminifera ANT-2200 was digested with BamHI+KpnI to clone the lux gene cluster.
  • Genomic DNA of P.luminescens DSM15139 was digested with XbaI to clone plu3535-plu3532; digested with XbaI+XmaI to clone plu2670.
  • the genomic DNA of Streptomyces albicans S. albus was digested with EcoRV or Cas9-gRNA to clone the salinomycin gene cluster.
  • the genomic DNA of mouse cell B 16 was digested with HpaI to clone Prkar1a, digested with BamHI+KpnI to clone Dpy30, and digested with SwaI to clone Wnt4 or Lmbr1l-Tuba1a.
  • Human genomic DNA was digested with NdeI+BstZ17I to clone IGFLR1-LIN37, digested with SpeI to clone DPY30, digested with BstZ17I to clone IGFLR1-ARHGAP33, and digested with NdeI to clone ZBTB32-LIN37.
  • the digested genome was extracted with phenol chloroform and ethanol precipitated, and finally dissolved in ddH 2 O at a concentration of about 1 ug/ul.
  • S. pyogenes Cas9 protein was purchased from New England Biolab. Cas9 digestion of S. albus genomic DNA of Streptomyces cerevisiae was carried out in an 800 ul reaction system containing 80 ul of 10 ⁇ Cas9 reaction buffer (NEB) (NEB), 80 ⁇ g of genomic DNA, 40 ⁇ g of gRNA-2, 40 ⁇ g of gRNA-7 and 20 ⁇ g of Cas9. . Since the cleavage efficiency of Cas9 is severely affected by the purity of the DNA substrate, in this assay, S. albus genomic DNA of S. albicans needs to be extracted with phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0).
  • PCR amplification (Fig. 4)
  • the template plasmid of the p15A-em vector was p15A-Pamp-luxABECD (purchased from Genebridge), using PrimeSTAR Max DNA polymerase (Takara), and the primers used (Table 1) contained 80 nucleotides. The homology arms were purified by PAGE. The PCR product was subjected to gel recovery to exclude interference from the primers to subsequent experiments.
  • the kit used was QIAquick Gel Extraction Kit (Qiagen), and finally the DNA was eluted with ddH 2 O at a concentration of approximately 200 ng/ul, and 200 ng was used for ExoCET cloning experiments.
  • the construction method of the pBeloBAC11 vector for cloning the 106 kb salinomycin gene cluster and the pBAC2015 vector for cloning plu3535-3532 was as described in the literature 43 and the literature 46 .
  • the homologous arms at both ends were digested by BamHI, and then the DNA was collected by phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0) and isopropanol precipitation, and finally dissolved in ddH 2 O, concentration. About 1 ug/ul.
  • ExoCET cloning experiments were performed using 1 ⁇ g BAC vector.
  • the lowercase letter is the homologous arm sequence.
  • PCR products amplified with PrimeSTAR Max DNA polymerase carry a 40 bp homology arm between each other.
  • the PCR product was purified by gel recovery using a QIAquick Gel Extraction Kit (Qiagen) and finally eluted with ddH 2 O at a concentration of approximately 200 ng/ul. Each fragment was assembled using 250 ng.
  • the mVenus-PGK-neo element was PCR amplified using PrimeSTAR Max DNA polymerase (Takara) using pR6K-2Ty1-2PreS-mVenus-Biotin-PGK-em7-neo 17 as a template.
  • the primers used are shown in Table S7.
  • the PCR product was purified by kit QIAquick PCR Purification Kit (Qiagen) using ddH 2 O at a concentration of approximately 100 ng ul -1 . 200 ng was used for homologous recombination experiments.
  • T5 exo-enzyme T5 exo
  • T7 exonuclease T7 exo
  • Klenow large fragment kle
  • T7 DNA polymerase T7pol
  • Lambda exonuclease Lam exo
  • exonuclease III Exo III
  • Gibson assembly NEB, cat.E2611
  • the cells were repeatedly centrifuged, resuspended, and centrifuged again, and the cells were suspended with 20 ul of ice ddH 2 O. 5 ul of desalted in vitro assembled product was added, while in the mVenus-PGK-neo element insertion assay a mixture of 200 ng of plasmid and 200 ng of PCR product was added.
  • the mixture of cells and DNA was transferred to a 1 mm galvanic cup, and an electric shock was performed with an Eppendorf electroporator 2510, a voltage of 1350 V, a capacitance of 10 uF, and a resistance of 600 ⁇ .
  • LB 1 ml of LB was added to the pulsing cup, the cells were washed and transferred to a 1.5 ml tube in a well, placed on an Eppendorf thermomixer at 37 degrees, and incubated at 950 rpm for 1 h. Finally, an appropriate amount of the bacterial solution was applied to an LB plate supplemented with a suitable antibiotic (15 ug/ml chloramphenicol or 15 ug/ml kanamycin) and cultured at 37 degrees overnight.
  • a suitable antibiotic 15 ug/ml chloramphenicol or 15 ug/ml kanamycin
  • Example 1 The combination of in vitro assembly and full length RecE/RecT increases the efficiency of direct cloning
  • the sequence of the genomic fragment of the gene cluster is identical (homologous arm), and the linear vector is homologously recombined with the genomic DNA fragment of the target by the homologous arms at both ends to form the final circular plasmid.
  • the cloning vector and BamHI+KpnI digested genomic DNA were first treated in vitro with different exonucleases, and then the in vitro reaction product was transformed into E. coli cells expressing the RecE/RecT recombinase.
  • the exonucleases tested include: T4 DNA polymerase (T4pol), Klenow fragment of DNA polymerase I (Kleow fragment of DNA Polymerase I; Kle), T7 DNA polymerase (T7pol), nucleic acid Exonuclease III (ExoIII); Phusion DNA polymerase (Phusion DNA polymerase; Phu), T5 exonuclease (T5exo); T7 exonuclease (T7exo) and Lambda nucleic acid Lambda exonuclease ( ⁇ exo) (Fig. 2a).
  • T4pol T4 DNA polymerase
  • Kle Klenow fragment of DNA polymerase I
  • T7pol T7 DNA polymerase
  • ExoIII nucleic acid Exonuclease III
  • Phusion DNA polymerase Phusion DNA polymerase
  • Phu Phu
  • T7exo T7 exonuclease
  • Fig. 2a Lambd
  • the inventors compared the direct cloning efficiency of the full-length RecE/RecT alone, T4pol in vitro alone, and T4pol in vitro annealing and full length RecE/RecT, respectively (Fig. 1f).
  • RecE/RecT was used alone, the 14 kb lux gene cluster was also cloned directly from the luminescent bacterium chromosome on the p15A vector with a high degree of accuracy (427).
  • direct cloning of T4pol in vitro assembled reaction products into a standard E. coli engineered bacterium was much more efficient (427 vs 4,880).
  • E.coli endogenous DNA repair system can skillfully close the plasmid backbone; (2) T4pol is highly efficient in in vitro assembly (4880); (3) T4pol is assembled in vitro and RecE/RecT cells ExoCET technology combined with internal recombination is more efficient than any of these technologies (32500).
  • At least one homology arm is placed at the end.
  • another homology arm can be placed inside the DNA fragment of interest because RecE/RecT can localize it and recombine it. This is very advantageous for the use of direct cloning to construct expression vectors, since one of the homology arms can be placed at the very end of the 3' end of the target fragment, and the 5' end of the gene of interest can be directly placed in the promoter and ribosome binding using internal homologous arms. Under the control of the locus.
  • RecT is a single-stranded DNA annealing protein
  • RecA has a certain improvement in the efficiency of direct cloning.
  • Table 2 Large fragments of genomic DNA cloned directly from bacteria, mammalian cells, and human blood using ExoCET.
  • the inventors had previously attempted to directly clone the 106 kb salinomycin gene cluster from the Streptomyces albus genome, so the inventors had to divide it into three fragments and then clone it. Stitch into a complete gene cluster 43 .
  • ExoCET using a BAC vector with a homology arm and EcoRV-cleaved genomic DNA, the inventors can directly clone the 106 kb salinomycin gene cluster into the BAC vector, and obtain a correct rate of 2/24. (Table 2 and Figure 6).
  • ExoCET has a significantly superior performance in direct cloning of large DNA fragments compared to RecET.
  • Gibson assembly uses T5exo, Phusion DNA polymerase and Taq DNA ligase to assemble DNA molecules with homology arms in between.
  • ExoCET can't assemble more than 13 DNA fragments
  • Gibson can't assemble more than 16 DNA fragments
  • Gibson+ETgA can assemble at least 20 DNA fragments into a 54.9 kb plasmid. Therefore, combining in vitro assembly with RecET recombination, the advantages for DNA assembly are obvious.
  • ExoCET can also be used to directly clone DNA fragments from mammalian genomes including blood, disease-associated cell lines, etc. to facilitate haplotype studies of SNPs and to rapidly construct haplotypes for nuclease-mediated human stem cell targeting.
  • Gene (HIT) targeting vector The importance of human stem cells isolated from patients, cord blood or somatic cell reprogramming in biomedical research has received increasing attention, and research on the precise modification of stem cell genome has received widespread attention. Transforming the human genome is more challenging than structuring the genome of experimental mice because human genetic diversity is complex. Syngeneic (sequence similarity) the importance of homologous recombination many years ago people mouse embryonic stem cells in gene targeting when he realized 14.
  • ExoCET is an effective way to quickly obtain ideal homology arms. Unlike the method of amplifying homology arms from the genome by PCR, ExoCET is not limited by fragment size, does not introduce mutations, and is capable of maintaining a haplotype of DNA.
  • the ends of the homology arms can also be selected according to the manner of genotyping (such as Southern hybridization or ligation PCR), so the length of the homology arms can be optimized.
  • ExoCET therefore offers advantages for individualized genomic surgery, especially when combined with CRISPR/Cas9 15 .
  • the inventors used ExoCET to construct isogenic targeting vectors to engineer mammalian genomes. Given the experience in studies of mouse embryonic stem cells 50, the inventors aimed from human or mouse genome directly cloned DNA fragments with 5 to 10kb as homologous arm ( Figure 8a and Figure 9a). It is noteworthy that these DNA fragments are not only the same gene but also maintain the polymorphic haplotype, so the inventors call it the "HIT" (haplotypic isogenic targeting) vector.
  • the ExoCET inventors directly cloned 8 to 9 kb DNA fragments (Fig. 8b and Fig.
  • ExoCET can also be used as the most reliable method for genotyping a modified genome, while Southern hybridization and ligation PCR produce false positive signals. Since long range PCR is prone to false positive signals in mammalian genotyping studies, the inventors previously wanted to target mouse embryonic stem cells targeting Kmt2d-AID-neo by long fragment PCR by Southern hybridization. Undergo verification. However, the inventors never get a good probe. Therefore, the inventor uses the ExoCET method shown in Fig. 10a, A DNA fragment containing the entire targeting element was cloned from the genome of four possible Kmt2d-AID-neo target mouse embryonic stem cells.
  • the inventors previously failed to determine whether a Klf4-Venus-neo-targeted mouse embryonic stem cell was successfully targeted, as long-segment PCR and Southern hybridization have not been able to obtain an exact signal.
  • the inventors did not clone a DNA fragment having kanamycin resistance in the corresponding region on the genome (Table 3). Restriction analysis of the cloned chloramphenicol resistant plasmid revealed that 50% of them contained the wild type DNA sequence (Fig. 11). Therefore, the inventors know exactly that this cell has not been correctly targeted.
  • Table 4 Optimize the amount of Oct4-Venus-neo targeted mouse embryonic stem cell genomic DNA required for ExoCET genotyping
  • ExoCET genotyping does not produce a false positive signal compared to long fragment PCR. Compared with Southern hybridization, the operation is simpler and does not require a cumbersome screening hybridization probe process.
  • ExoCET genotyping restriction enzyme sites for the release of intact targeting elements are readily available, and in the case of well-prepared genomes, genotyping results are obtained in three days. More importantly, ExoCET never produces a false positive signal. Since the targeting element has a selection marker, as long as 500 ng of restriction enzyme genomic DNA is sufficient to obtain better cloning efficiency (Table 4). To increase the throughput of ExoCET genotyping, cells cultured in 96-well plates can be used.
  • the inventors can clone a DNA fragment of up to 50 kb from a genome of 3.0 x 109 bp.
  • the inventors diluted 1 ng of the genomic DNA of Bacillus brevis to 10 ⁇ g of Bacillus subtilis genomic DNA to mimic the metagenomics, successfully cloned the 14 kb lux gene cluster by ExoCET, and obtained considerable efficiency (Table 5).
  • Environmental samples typically contain more than 10 4 species 51-53 , so the results can motivate the inventors to apply ExoCET cloning techniques to metagenomic samples.
  • Table 5 The 14 kb lux gene cluster was cloned directly from the diluted B. luminiferi genome by ExoCET.
  • Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821 (2012).

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Abstract

一种同源重组的方法，所述方法包括使用第一核酸外切酶处理两个或两个以上目标核酸分子，然后使处理后的目标核酸分子在第二核酸外切酶和退火蛋白的存在下进行同源重组，其中发生同源重组的目标核酸分子之间共享至少一段序列同源区域。还提供了用于上述方法的试剂盒。

Description

基因组大片段直接克隆和DNA多分子组装新技术 技术领域
本发明涉及核酸克隆和组装方法，更具体涉及脱氧核糖核酸(DNA)克隆和组装的新方法。
发明背景
DNA克隆是分子生物学和生物技术的核心内容，是进行基因功能研究的关键技术。随着DNA测序技术的不断进步和测序成本的不断降低，全基因组测序变得越来越容易。人们发现基因组中蕴藏着丰富的尚未开发的资源。短的DNA片段可以很容易通过PCR或者化学合成获得，但是克隆大于10kb的DNA片段传统方法则要依赖于DNA文库的构建和筛选。传统的文库构建和筛选的方法不但操作复杂、耗时、费力，而且目的DNA片段经常分散的位于几个不同的克隆上，在进行基因功能研究时往往需要对其进行亚克隆，删除多余序列或者需要将其缝合成一个完整地生物合成途径，因此传统的文库构建和筛选的方法已经不能满足时代发展的需要，研究者们迫切需要简单、高效和快捷的基因组DNA克隆和修饰技术。随着合成生物学技术的进步，大于10kb的DNA大片段还可以通过Gibson体外组装1或者DNA assembler体内组装2等方式由小片段组装而成。但是上述DNA组装需要PCR或者化学合成来制备小片段，容易引入随机突变并为后期基因功能研究带来不便。同时，上述方式对于要组装的DNA片段的量要求较高，需要采用纯度和浓度相对较高的目标DNA片段，不能用于直接从酶解的基因组DNA片段混合物中组装目的DNA片段。
直接从基因组DNA中将特定的DNA区域克隆到载体中，在本文中称为“直接克隆”。RecET直接克隆技术的原理是：Rac噬菌体重组蛋白——全长的RecE和RecT能够在大肠杆菌细胞内高效地介导线性DNA分子之间发生同源重组——线线重组。其中RecE是5’-3’核酸外切酶，RecT是单链DNA退火蛋白，RecE和RecT之间的蛋白-蛋白相互作用是线线重组所必须的，RecET联合作用的线线重组效率是单独作用的1000倍。RecET直接克隆技术能将大于10kb的基因组DNA片段直接捕获到表达载体上，而且还能实现2～5个DNA片段的组装。在基因组上一般都能找到合适的限制性酶切位点将目的DNA片段释放 出来，然后将限制性酶切的基因组DNA和线性载体通过电转化导入表达了RecET重组酶的大肠杆菌细胞内，在细胞内目的DNA片段和线性载体通过两端的同源臂发生同源重组并形成环状质粒，最后通过抗生素筛选和限制性酶切分析可以获得含有重组DNA分子的菌落。在线性载体和基因组DNA都制备好了的情况下，完成目的DNA片段的直接克隆只需要3天的时间。目前该技术已被广泛应用于细菌天然产物生物合成途径的克隆和异源表达研究。比如来源于发光杆菌发光光杆状菌的10个10-52kb的聚酮合酶(PKS)/非核糖体多肽合成酶(NRPS)基因簇3，来源于类芽孢杆菌Paenibacillus larvae中的12kb sevadicin基因簇4，来源于丁香假单胞菌Pseudomonas syringae中的19kb syringolin基因簇5，来源于伯克氏菌Burkholderia DSM7029中的25kb glidobactin基因簇6，以及来源于大肠杆菌E.coli Nissle 1917中的50kb colibactin基因簇7。
与文库构建和筛选相比，直接克隆所具有的优势驱动了该领域的很多相关研究。Larionov等人建立了依靠酵母重组系统的TAR克隆技术8，然后他们将TAR与Cas9切割结合起来提高了TAR克隆的效率使其应用起来更方便9，10。最近建立的CATCH(Cas9-Assisted Targeting of Chromosome Segments)技术利用Cas9体外切割和Gibson组装将DNA大片段克隆到BAC上11。该方法目前只被应用于原核生物基因组，而且在对重组DNA进行限制性酶切鉴定之前需要先对菌落进行PCR预筛选。据发明人所知包括上述两种方法在内的所有目前已知的直接克隆方法都局限于某些专家才能操作。
尽管RecET直接克隆技术在从细菌基因组上克隆50kb以下的DNA片段时操作方便3-6，12，但是却很难从细菌基因组上克隆更大的DNA片段，也无法从哺乳动物基因组上克隆DNA片段。这是由于RecET直接克隆技术依赖于细胞内表达的RecET重组酶，只有当克隆载体和目的DNA片段同时进入一个大肠杆菌细胞内并相遇才能发生同源重组，这就导致了该技术的局限性：(1)受到基因组DNA片段大小的限制，比如无法从从细菌基因组中克隆大于50kb的DNA片段或者从哺乳动物(比如老鼠和人)中克隆大于10kb的基因组片段。这是因为某一段DNA在基因组中的浓度是很低的，这就导致了目的DNA片段与克隆载体在转化时同时进入一个细胞的概率是很小的。而且由 于受到基因组DNA制备过程中物理剪切力的影响的影响，要克隆的目的DNA片段越大，其在基因组中的浓度就越低，最终导致目的DNA片段与克隆载体共同进入一个细胞的几率也就越低。老鼠(2800Mb)和人(3200Mb)的基因组比细菌基因组(5Mb)大了将近3个数量级，所以相同大小的基因组DNA片段在哺乳动物基因组中的浓度要比细菌低得多，因此在进行哺乳动物基因组DNA片段直接克隆时，目的DNA片段与克隆载体同时进入一个大肠杆菌细胞的几率也低得多。(2)受到DNA片段数目的影响，对于多片段组装来说，当DNA片段数量超过4个以上时，组装效率急剧降低，目前RecET直接克隆技术最多只能组装5个DNA片段。对于多片段组装来说，DNA片段的数量越多，它们共同进入一个细胞的几率就越低，因此组装效率也就越低。因此业界特别需要一个不受DNA片段大小和基因组复杂性限制的易于操作而又应用广泛的直接克隆技术。
为了解决从基因组中直接克隆大片段的技术问题，发明人在体外先用核酸外切酶处理基因组DNA和克隆载体，然后再将体外反应产物在RecET重组酶的存在下进行同源重组，从而建立了ExoCET克隆技术。ExoCET技术不但能够从细菌基因组上直接克隆＞100kb的DNA片段，而且能够从哺乳动物细胞和人血中克隆＞50kb的DNA片段。ExoCET技术还能高效的组装至少20个以上的DNA片段，使其形成一个完整的质粒。
与RecET直接克隆技术一样，ExoCET与PCR相比的优势在于保真性高，不会破坏DNA的单倍型，而且目的DNA直接克隆到质粒载体上以利于表达研究。此外，ExoCET比Gibson组装更高效，因为Gibson依赖于体外组装产生的环状DNA分子。而且，Gibson组装在直接克隆中会产生很严重的由空载体自身环化产生的背景，这也许是为什么Jiang等人11在对重组DNA进行鉴定之前要进行PCR预筛选的原因；也是Zhou等人13在利用Gibson组装从链霉菌Streptomyces conglobatus基因组上克隆要通过conglobatin基因簇时，需要通过琼脂糖凝胶电泳去除限制性酶切产生的20kb以下的基因组DNA片段的原因。
ExoCET还可以用于从包括血液，疾病相关细胞系等来源的哺乳动物基因组上直接克隆DNA片段以助于SNPs的单倍型研究以及为核酸酶介导的人类干细胞打靶来快速构建单倍型同基因(HIT)打靶载体。 从病人、脐带血或体细胞重编程分离到的人类干细胞在生物医学研究的重要性越来越受到人们的重视，对干细胞基因组的精确修饰方法的研究也受到人们的广泛关注。改造人类基因组比改造实验鼠的基因组要更有挑战性，因为人类的遗传多样性很复杂。同基因性(序列相似性)对同源重组的重要性在很多年前人们对老鼠胚胎干细胞进行基因打靶的时候就意识到了14。但是，序列错配对同源重组的影响还未研究清楚，比如单个错配(一个SNP或一个indel)到底能降低多少重组效率？错配距离重组位点的远近是如何影响重组效率的？多个错配是如何影响重组效率的？这些问题目前都还没有搞清楚。无论如何，在基因打靶中使用完全相同的序列很明显是大家极力推荐的，因此ExoCET是一种快速获得理想同源臂的有效方式。与通过PCR从基因组上扩增同源臂的的方法不同，ExoCET不受片段大小的限制，不会引入突变，而且能够维持DNA的单倍型。此外同源臂的末端还可以根据基因分型的方式(比如Southern杂交或者连接PCR)进行选择，所以可以对同源臂的长度进行优化。因此ExoCET为个体化基因组手术提供了优势，尤其是当其与CRISPR/Cas915联合起来时。ExoCET还可以作为一种对修饰后的基因组进行基因分型的最可靠方法，而Southern杂交和连接PCR会产生假阳性信号。
ExoCET具有的从复杂基因组上选择性捕获DNA大片段的能力使其成为疾病诊断和病理学检验的手段，比如为个体化医疗直接捕获DNA序列或者从病人样本中分离DNA病毒。ExoCET将会广泛应用于功能基因组学和比较基因组学研究中，尤其是直接克隆原核生物合成途径或者为合成生物学组装多个DNA分子。
发明内容
在一个方面，本发明提供了一种同源重组的方法，所述方法包括使用第一核酸外切酶处理两个或两个以上目标核酸分子，然后使处理后的目标核酸分子在第二核酸外切酶和退火蛋白的存在下进行同源重组，其中发生同源重组的目标核酸分子之间共享至少一段序列同源区域。
在一个方面，本发明提供了一种同源重组的方法，其中所述方法包括使用第一核酸外切酶处理第一核酸分子与第二核酸分子，然后使处理后的第一核酸分子与第二核酸分子在第二核酸外切酶和退火蛋白 的存在下进行同源重组，其中第一核酸分子和第二核酸分子之间共享至少一段序列同源区域。
在一个方面，本发明提供了一种组装线性核酸分子的方法，所述方法包括使用第一核酸外切酶处理两个或两个以上的核酸分子，然后使处理后的核酸分子混合物在第二核酸外切酶和退火蛋白的存在下进行同源重组，其中每个核酸分子与在所产生的组装产物中形成邻近物的核酸分子共享至少一段序列同源区域。
在一个方面，本发明提供了一种克隆基因组DNA的方法，其中方法包括使用第一核酸外切酶处理基因组DNA片段混合物和线性克隆载体，然后使处理后的基因组DNA片段混合物中的目标DNA片段和线性克隆载体在第二核酸外切酶和退火蛋白的存在下进行同源重组，其中基因组DNA片段混合物中目标DNA片段与线性克隆载体之间共享至少一段序列同源区域。
根据前述方面任一项的方法，其中所述序列同源区域可以在目标核酸分子内部或末端，优选至少一个同源区域在目标核酸分子末端，更优选同源区域均在目标核酸分子末端。
根据权利要求前述方面任一项的方法，其中所述同源区域的长度为至少6、至少10、至少20、至少25、至少30、至少40、至少50、至少60、至少70、至少80个核苷酸，优选25个、40个或80个，最优选80个核苷酸。
根据前述方面任一项的方法，其中所述第一核酸外切酶可以是5’到3’核酸外切酶或是3’到5’核酸外切酶，优选T4 DNA聚合酶、DNA聚合酶I的Klenow片段、T5核酸外切酶、T7核酸外切酶，最优选T4 DNA聚合酶或T5核酸外切酶。
根据前述方面任一项的方法，其中所述第一核酸外切酶处理包括体外连接步骤，所述体外连接步骤使两个或两个以上目标核酸分子或者所述第一核酸分子与所述第二核酸分子连接起来，或者使处理后的线性克隆载体和基因组DNA片段混合物中的目标DNA片段连接起来。
根据前述方面任一项的方法，其中所述第一核酸外切酶处理包括酶切和退火步骤，其中不同核酸分子的酶切可以是单独进行也可以是同时进行，诸如在单个样品中的混合物中。
根据前述方面任一项的方法，其中所述第一核酸外切酶处理还包 括加入DNA聚合酶，dNTP和DNA连接酶的步骤。
根据前述方面任一项的方法，其中所述第一核酸外切酶处理还包括加入具有3′-5′核酸外切酶活性的DNA聚合酶。
根据前述方面任一项的方法，其中所述第一核酸外切酶处理排除添加dNTPs。
根据前述方面任一项的方法，其中所述第一核酸外切酶处理是T4DNA聚合酶处理或Gibson组装。
根据前述方面任一项的方法，其中所述第二核酸外切酶是RecE，优选地，所述RecE是重组表达的。
根据前述方面任一项的方法，其中所述单链退火蛋白包括RecA、RAD51、Redβ、RecT、Pluβ或RAD52，优选其中所述退火蛋白是RecT，更优选地，所述RecT是重组表达的。
根据前述方面任一项的方法，其中所述退火蛋白是RecT，优选地，所述RecT是重组表达的。
根据前述方面任一项的方法，其中所述同源重组在体外或宿主细胞中进行。
根据前述方面任一项的方法，其中所述宿主细胞可以是酵母细胞，优选酵母细胞是酿酒酵母细胞；或者是细菌细胞，优选细菌细胞是枯草芽孢杆菌或大肠杆菌。
根据前述方面任一项的方法，其中所述宿主细胞表达核酸外切酶，优选第二核酸外切酶、和退火蛋白。
根据前述方面任一项的方法，其中所述其中所述宿主细胞表达核酸外切酶、退火蛋白和Redγ，优选地，所述宿主细胞还表达RecA，最优选地，所述宿主细胞表达RecE、RecT、Redγ和RecA。
根据前述方面任一项的方法，其中所述宿主细胞是表达全长RecE和RecT的大肠杆菌细胞，优选地，所述宿主细胞是表达全长RecE、RecT和Redγ的大肠杆菌细胞，最优选地，所述宿主细胞是表达全长RecE、RecT、Redγ和RecA的大肠杆菌细胞。
根据前述方面任一项的方法，其中所述宿主细胞是表达截短的RecE和RecT的大肠杆菌细胞。
根据前述方面任一项的方法，其中所述宿主细胞是表达Redα和Redβ的大肠杆菌细胞。
根据根据前述方面任一项的方法，其中所述宿主细胞通过在质粒载体和/或染色体上表达核酸外切酶，优选第二核酸外切酶、退火蛋白、Redγ和/或RecA，优选地，通过质粒载体表达，最优选地，通过质粒载体和染色体同时表达。
根据前述方面任一项的方法，其中所述目标核酸分子或目标DNA片段是线性的，优选选自用核酸内切酶切割的DNA片段、PCR扩增的DNA片段、基因组DNA片段、cDNA文库成员、源自BAC的片段和克隆载体片段。
根据前述方面任一项的方法，其中所述核酸内切酶可以是限制性内切酶或可编程的核酸内切酶，例如Cas9。
根据前述方面任一项的方法，其中所述目标核酸分子或DNA片段的个数是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个或更多个。
根据前述方面任一项的方法，其中所述目标核酸分子包括长度为0.5kb或更长(例如，1kb或更长、2.5kb或更长、4kb或更长、5kb或更长、7.5kb或更长、10kb或更长、15kb或更长、20kb或更长、25kb或更长、40kb或更长、50kb或更长、75kb或更长或100kb或更长)的序列。
根据前述方面任一项的方法，其中：
-所述两种或更多种目标核酸分子、第一目标核酸分子和第二目标核酸分子或目标DNA片段包括一种或多种PCR扩增的DNA片段、基因组DNA片段、cDNA文库成员、和/或衍生自BAC的片段；
-所述第一核酸外切酶具有3′-5′核酸外切酶活性，优选是T4 DNA聚合酶；
-所述核酸外切酶处理在体外在不存在dNTPs的情况下进行；
-所述第二核酸外切酶是全长RecE；
-所述退火蛋白是RecT；和
-所述同源重组在表达所述全长RecE和RecT的细菌宿主细胞中进行，优选大肠杆菌。
根据前述方面任一项的方法，其中：
-所述两种或更多种目标核酸分子包括一种或多种PCR扩增的DNA片段、基因组DNA片段、cDNA文库成员、和/或衍生自BAC的 片段，线性质粒和/或克隆载体片段，优选三种或更多种线性质粒和/或克隆载体片段；
-所述第一核酸外切酶包括Gibson组装；
-所述第二核酸外切酶是全长RecE；
-所述退火蛋白是RecT；和
-所述同源重组在表达所述全长RecE和RecT的细菌宿主细胞中进行，优选大肠杆菌。
根据前述方面任一项的方法，其中：
-所述两种或更多种目标核酸分子包括三种或多种PCR扩增的DNA片段、基因组DNA片段、cDNA文库成员、或衍生自BAC、线性质粒和/或克隆载体的片段，优选三种或更多种线性质粒和/或克隆载体片段；
-所述第一核酸外切酶处理包括Gibson组装；
-所述第二核酸外切酶是全长RecE；
-所述退火蛋白是RecT；和
-所述同源重组在表达所述全长RecE和RecT的细菌宿主细胞中进行，优选大肠杆菌。
试剂盒，其包括前述任一项的方法中所述的第一核酸外切酶和第二核酸外切酶或编码前述任一项的方法中所述第一核酸外切酶和第二核酸外切酶的核酸。
试剂盒，其包括前述任一项的方法中所述的第一核酸外切酶和第二核酸外切酶或编码所述第一核酸外切酶和第二核酸外切酶的核酸，优选地，所述试剂盒还包括表达第二核酸外切酶的宿主细胞，优选地，所述宿主细胞表达核酸外切酶、退火蛋白和Redγ，优选地，所述宿主细胞还表达RecA，最优选地，所述宿主细胞表达RecE、RecT、Redγ和RecA，所述宿主细胞可以是酵母细胞，优选酵母细胞是酿酒酵母细胞；或者是细菌细胞，优选细菌细胞是枯草芽孢杆菌或大肠杆菌，所述宿主细胞通过在质粒载体和/或染色体上表达核酸外切酶、退火蛋白、Redγ和/或RecA，优选地，通过质粒载体表达，最优选地，通过质粒载体和染色体同时表达，进一步优选地，所述试剂盒还可包括一个或多个预先制备的线性载体。
根据前述任一项的试剂盒，其中所述第一核酸外切酶是具有3′-5′ 核酸外切酶活性的DNA聚合酶，诸如T4 DNA聚合酶、DNA聚合酶I的Klenow片段、T5核酸外切酶或T7核酸外切酶，所述第二核酸外切酶是全长RecE。
根据前述任一项的试剂盒，其中所述试剂盒还包括表达第二核酸外切酶的宿主细胞，优选地所述宿主细胞中包含编码全长RecE、RecT、Redγ和RecA的核酸。
根据前述任一项的试剂盒，其中所述试剂盒还可包括一个或多个预先制备的线性载体。
根据前述任一项的方法或试剂盒在构建打靶载体中的用途。
根据前述任一项的方法或试剂盒在对哺乳动物细胞进行基因分型中的用途。
根据前述任一项的方法或试剂盒在合成DNA中的用途。
附图说明
图1.联合使用体外组装和RecE/RecT促进了直接克隆的效率。(a)从明亮发光杆菌(P.phosphoreum)ANT-2200基因组上直接克隆14kb lux基因簇的示意图。p 15A-cm载体和目的基因组DNA片段在最末端具有相同序列。(b)同源臂越长克隆效率越高。具有25bp，40bp或80bp同源臂的线性载体分别与基因组DNA混合，并且在退火和转化到阿拉伯糖诱导的E.coli GB05-dir之前用0.02U μl-1的T4pol在25℃下反应20分钟。误差线，s.d.；n＝3.(c)优化T4pol的浓度。具有80bp同源臂的线性载体与基因组DNA采用与(b)中相同的方式进行处理，唯一不同的T4pol的浓度。(d)T4pol的温育时间对克隆效率的影响。与(c)相同，使用0.02U μl-1的T4pol，但是采用不同的温育时间。(e)ETgA拷贝数越高克隆效率越高。与(d)相同，温育时间为1小时，然后将体外组装产物分别电转化到阿拉伯糖诱导的E.coli GB05-dir(在染色体上带有1个ETgA的拷贝)，含有pSC101-BAD-ETgA-tet的GB2005(在pSC101质粒上带有～5个ETgA的拷贝)或者含有pSC101-BAD-ETgA-tet的GB05-dir(带有～6个ETgA的拷贝)。(f)ExoCET提高了直接克隆的效率。与(e)相同，使用含有pSC101-BAD-ETgA-tet的GB05-dir(ExoCET)，或者不用T4pol处理(ETgA)，或者不用阿拉伯糖诱导(T4pol)。
图2.不同核酸外切酶对lux基因簇直接克隆的影响。p15A-cm载 体和明亮发光杆菌基因组DNA用核酸外切酶处理、退火后电转化到阿拉伯糖诱导的E.coli GB05-dir中。(a)初始检测不同的核酸外切酶。(b-d)优化Kle，T5exo和T7exo的浓度。(e)用最优浓度的T4pol，Kle，T5exo和T7exo酶切20min后的克隆效率比较。(f)优化T4pol的酶切温度和时间(0.02U μl-1)。误差线，s.d.；n＝3。
图3.退火速率对lux基因簇直接克隆的影响.误差线，s.d.；n＝3.p15A-cm载体和明亮发光杆菌基因组DNA用0.02U μl-1T4pol酶切之后采用不同的方式(A，B，C)退火之后电转化到阿拉伯糖诱导的E.coli GB05-dir中。
图4.线性克隆载体的制备和ExoCET直接克隆技术的流程图。(a)通过PCR扩增制备p15A-cm载体，所用引物带有80个核苷酸的同源臂。(b)ExoCET直接克隆的标准策略。体外组装产物转化到阿拉伯糖诱导的含有pSC101-BAD-ETgA-tet的GB05-dir中，通过抗生素筛选获得正确的重组子。
图5.(a)p15A-cm和14kb lux基因组DNA片段之间的80bp同源臂的位置：(A)两个同源臂都位于最末端；(B，C)一个位于距离末端1kb的位置，另一个位于最末端；(D)都位于距离末端1kb的位置。反应条件与图1f相同。(b)利用上述四种同源臂通过ETgA，T4pol或ExoCET获得到菌落数。(c)在GB2005中利用ExoCET和末端同源臂通过pSC101质粒表达的不同重组蛋白组合获得的14kb lux基因簇直接克隆效率：ETg-不表达RecA；Eg-不表达RecA和RecT；Tg-不表达RecA和RecE；pSC101-tet-空载体。误差线，s.d.；n＝3.
图6.利用EcoRV或Cas9酶切的白色链霉菌S.albus基因组DNA直接克隆106kb的盐霉素基因簇。(a)利用ExoCET从EcoRV或Cas9gRNA2/Cas9-gRNA7酶切的基因组DNA上将盐霉素基因簇克隆到pBeloBAC11载体上。同源臂(蓝色)先被插入BAC载体上并通过BamHI酶切将BAC载体线性化作为直接克隆载体。同源臂长度标示于基因组DNA片段的末端。(b)重组DNA的PvuII限制性酶切分析，正确的克隆用箭头标示。
图7 ExoCET和Gibson组装的效率比较.(a)从老鼠基因组上利用末端同源臂直接克隆含有Wnt4基因的45kb DNA片段的示意图。(b)通过ExoCET和Gibson方法获得的菌落数。p15A-cm和SwaI酶切的 老鼠基因组DNA分别通过ExoCET和Gibson处理，然后转化到阿拉伯糖诱导(ExoCET或Gibson+ETgA)或不诱导(T4pol或Gibson)的含有pSC101-BAD-ETgA-tet的GB05-dir中。(c)通过T4pol、ExoCET、Gibson和Gibson+ETgA将多个DNA片段组装成质粒的示意图。DNA片段大小为1.0kb～5.4kb，组装成的p5A质粒大小为29.8kb～54.9kb，而且带有氯霉素抗性(cm)。(d)多片段组装实验得到的克隆数及正确率。体外组装的产物转化到阿拉伯糖诱导(ExoCET或Gibson+ETgA)或不诱导(T4pol或Gibson)的含有pSC101-BAD-ETgA-tet的GB05-dir中。
图8.利用哺乳动物基因组DNA为DPY30构建HIT(单倍型同基因打靶)载体。(a)利用SpeI酶切的人类基因组DNA克隆DPY30终止密码子区域的示意图。直接克隆完成后，通过Redαβ重组工程mVenus元件16，17标记到DPY30的C端。(b)对利用从人血中分离的基因组DNA通过ExoCET进行直接克隆获得的重组DNA进行EcoRI酶切分析。(c)对利用从人类胚胎肾脏293T细胞中分离的基因组DNA通过ExoCET进行直接克隆获得的重组DNA进行EcoRI酶切分析。(d)对通过Redαβ重组工程插入mVenus元件后获得的重组DNA进行PvuII酶切分析。获得的所有克隆都是正确的，其中11泳道是对照。正确的克隆用箭头标示。
图9.利用老鼠细胞基因组DNA为Dpy30构建HIT(单倍型同基因打靶)载体。(a)利用BamHI+KpnI酶切的老鼠基因组DNA克隆Dpy30终止密码子区域的示意图。直接克隆完成后，通过Redαβ重组工程mVenus元件16，17标记到Dpy30的C端。(b)对利用从老鼠黑色素瘤B16细胞中分离的基因组DNA通过ExoCET进行直接克隆获得的重组DNA进行EcoRI酶切分析。(c)对通过Redαβ重组工程插入mVenus元件后获得的重组DNA进行NheI酶切分析。获得的所有克隆都是正确的，其中11泳道是对照。正确的克隆用箭头标示。
图10利用ExoCET对哺乳动物细胞进行基因分型。(a)利用ExoCET进行基因分型的原理图。限制性酶切位点分别位于打靶元件的上游和下游。(b)利用ExoCET对具有卡那霉素抗性的Kmt2d-AID-neo打靶老鼠胚胎干细胞进行基因分型。利用SspI和SpeI从基因组上释放含有打靶元件的DNA片段。使用10ug酶切的基因组DNA和PCR扩 增的p15A-cm载体进行ExoCET克隆。克隆到p15A载体中的打靶片段和野生型片段可以通过双划线和限制性酶切区分开。
图11.对Klf4-Venus-neo打靶的老鼠胚胎干细胞进行ExoCET基因分型得到的氯霉素抗性菌落的EcoRV+PstI限制性酶切分析。
具体实施方案
DNA重组工程是在大肠杆菌(E.coli)细胞内利用噬菌体syn/exo蛋白(主要是Redα和Redβ)18-22介导的同源重组对DNA分子进行修饰的基因工程技术。DNA重组工程技术最开始是在E.colisbcA(recBC抑制子)菌株中发现的，该菌株具有高效介导带有同源臂(homology boxes)的DNA分子之间发生同源重组的活性23。sbcA菌株是由AJ Clark在E.coli中寻找同源重组途径的经典试验中发现的。他利用对DNA损伤非常敏感的recBC菌株来筛选其抑制子时发现了具有RecE和RecT表达活性的sbcA突变菌株24-26。随后研究发现RecE和RecT是由整合在染色体上的Rac噬菌体表达的，它们与噬菌体的Redα和Redβ的功能相同27，而且在sbcA突变菌株只表达了RecE蛋白C端的280个氨基酸28-30。截短的RecE与Redα(266个氨基酸)相似，是一个5’-3’核酸外切酶31，RecT与Redβ类似，是一个单链DNA退火蛋白(SSAP，single strand annealing protein)32。RecE/RecT和Redα/Redβ属于5’-3’核酸外切酶/SSAP syn/exo蛋白对21，33，而且每对蛋白之间特异性的蛋白-蛋白相互作用势双链DNA的同源重组所必需的29，34，35。Redα/Redβ介导的同源重组主要发生在复制叉上而且要求复制进行36，37。虽然一开始发明人是通过截短的RecE/RecT发现的重组工程技术，但是后来发明人主要利用Redα/Redβ来修饰DNA分子，因为后者的效率更高16，38-42。尽管如此，发明人还是在不停地研究RecE/RecT的特性，而且发现RecE N端的600个氨基酸残基将其重组活性由复制依赖型变成了复制不依赖型3。因此，两个线性DNA分子可以通过很短的同源臂发生高效的同源重组而形成环状质粒。与Redα/Redβ重组工程相比，这种线线重组机制具有不同的应用范围，比如从基因组上直接克隆DNA大片段3-6，12或者进行DNA的多片段组装15，43。
本发明提供了一种用于在共享至少一段序列同源区域的两个或两个以上的目标线性核酸分子之间进行同源重组(线线重组)的方法， 其中所述方法包括使用第一核酸外切酶处理目标线性核酸分子的混合物；然后使处理后的目标线性核酸分子在第二核酸外切酶和退火蛋白的存在下进行同源重组。其中所述第二核酸外切酶可以是RecE，来自大肠杆菌K12的全长RecE的氨基酸序列在WO2011/154927中公开。其中所述第二核酸外切酶也可以是截短的RecE，这些截短形式的RecE包括第588-866、595-866、602-866或606-866位氨基酸构成的RecE蛋白29。
所述第二核酸外切酶与退火蛋白共同介导同源重组。在一些实施方案中，在本发明的方法中使用的退火蛋白是WO2011/154927中公开的退火蛋白。优选的，所述退火蛋白是RecT或其片段(源自Rac噬菌体)。更优选的，所述退火蛋白是全长RecT并且所述第二核酸外切酶是全长RecE。但是，可以使用任何其它合适的退火蛋白，只要该退火蛋白与所使用的核酸外切酶共同作用。其它合适的噬菌体退火蛋白的实例提供于WO02/062988。线线重组可以在缺乏RecT表达的某些宿主细胞如大肠杆菌菌株GB2005中发生，可能是由于存在某些内源RecT-样活性。但是，由全长RecE介导的线线重组的效率在RecT的存在下显著升高。
本发明的方法在宿主细胞中可受到全部或部分的影响。合适的宿主细胞包括许多物种的细胞，包括寄生虫、原核生物和真核生物，但是细菌如革兰氏阴性细菌是优选的宿主。更优选的，宿主细胞是肠细菌细胞，如沙门氏菌(Salmonella)、克雷伯氏菌(Klebsiella)、芽孢杆菌(Bacillus)、奈瑟氏菌(Neisseria)、发光杆菌(Photorhabdus)或大肠杆菌(Escherichia coli)细胞(本发明的方法在已检测的所有大肠杆菌菌株中都起着有效的作用)。优选的宿主细胞是大肠杆菌K12。然而应当注意，本发明的方法同样适用于真核细胞或生物体中，如真菌、酵母、植物或动物细胞。该系统已经证实在小鼠ES细胞中具有功能，并且有理由推测在其他真核细胞细胞中也有功能。典型地，宿主细胞是分离的宿主细胞，但是同样可以使用未分离的宿主细胞。
本发明所述宿主细胞包含编码核酸外切酶(优选全长RecE)、退火蛋白(优选RecT)和Redγ的核酸。在一些实施方案中，所述宿主细胞还包含编码RecA的核酸。优选地，所述宿主细胞表达RecE、RecT和Redγ、以及任选的RecA。最优选的，所述宿主细胞表达RecE、 RecT、Redγ和RecA。
本发明所述核酸外切酶、退火蛋白、Redγ和/或RecA可以由外源DNA在宿主细胞中重组表达，例如，由转化进宿主细胞中的载体表达。一个合适载体的实例是pSC101质粒，但是也可以使用其它合适的载体。任何合适的启动子都可以用来驱动这些蛋白的表达。但是，在表达RecE时，优选诱导型启动子，如阿拉伯糖诱导型启动子(PBAD)或鼠李糖诱导启动子(PRhaSR)。这些启动子都是本领域公知的。
本发明所述宿主细胞通过在质粒载体或者染色体上由诱导型启动子表达核酸外切酶、退火蛋白、Redγ和/或RecA。优选地，核酸外切酶、退火蛋白、Redγ和/或RecA通过质粒载体在宿主细胞中表达。最优选地，核酸外切酶、退火蛋白、Redγ和/或RecA通过质粒载体和染色体同时在宿主细胞中表达。
大肠杆菌K12宿主细胞的基因组中包括全长recE基因和recT基因的内源性拷贝，它们存在于已整合到宿主基因组中的Rac噬菌体。但是，由于该基因是沉默的，因此全长RecE的表达并不能由该整合基因自然发生。因此，在5’到3’核酸外切酶由外源DNA表达的实施方案中，可在不存在内源性recE基因情况下实施所述方法。
还提供转化了编码如上所述核酸外切酶的核酸分子的宿主细胞。优选地，所述核酸外切酶由所述核酸分子表达，因此本发明还提供了表达本发明的方法中列举的核酸外切酶的宿主细胞。核酸外切酶优选地在诱导性启动子的控制下表达，如阿拉伯糖诱导型启动子(PBAD)或鼠李糖诱导型启动子(PRhaSR)。
还设想在前述实施方案中，本发明的方法在体外受到全部或者部分的影响。例如，可以使用纯化的5’到3’核酸外切酶和退火蛋白(优选纯化的RecE和RecT蛋白)，或者使用表达所述5’到3’核酸外切酶和退火蛋白的大肠杆菌细胞的提取物。当在体外实施该方法时，对所述第一和第二线性目标核酸分子进行预处理以暴露单链同源末端是有利的。
线线重组需要目标线性核酸分子之间必须共享至少一个序列同源区域，通过所述区域发生同源重组。在一些实施方案中，所述第一目标核酸分子与所述第二目标核酸分子共享一个序列同源区域，以使第一和第二目标核酸分子之间的线线重组产生线性产物。在所述第一和 第二线性核酸和一个或多个额外的线性核酸之间发生线线重组以形成线性产物的实施方案中，每个线性核酸与在线线重组反应的线性产物中形成其邻近物的线性核酸共享一个序列同源区域。在所述第一和第二线性核酸和一个或多个额外的线性目标核酸分子之间发生线线重组以形成环状产物的实施方案中，每个线性核酸与在线线重组反应的环状产物中形成其邻近物的线性核酸共享一个序列同源区域。在一些实施方案中，所述第一目标核酸分子和所述第二目标核酸分子共享两个序列同源区域，以使第一和第二目标核酸分子之间的线线重组形成环状分子。本领域技术人员知道如何设计同源区域以形成线性分子或环状分子。
优选的，至少一个同源臂在每个线性片段的最末端。当同源臂在每个线性片段的最末端并且不同的同源臂在另一端时，这些序列同源区域或“同源臂”产生最佳构型，如此构造这些同源臂以使重组产生环。当同源臂不位于末端时可以发生线线重组，但是效率会降低。因此，在优选的实施方案中，所述同源的至少一个区域位于所述目标核酸分子的一个或两个末端的最外端。在一些实施方案中，所述同源区域位于所述某一目标核酸分子的内部。
本发明所述的序列同源区域的长度为至少4、至少6、至少10、至少20、至少30个、至少40、至少50、至少60、至少70、至少80、至少90、至少100个核苷酸。例如，在一些实施方案中，序列同源区域为4-6、6-9、6-30、6-100、10-20、20-29、20-40、20-50、10-100、25-30、25-40、25-50、30-40、30-50、40-50、40-80个或80个以上核苷酸。同源重组的效率通常会随着使用的同源臂的长度而增加，因此可以使用较长的同源臂。
两个核酸分子之间的“同源”是指对两个核酸分子的序列进行比对时，有许多核苷酸残基在所述序列的相同位置上是相同的。同源的程度很容易计算出来(Computational Molecular Biology，Lesk，A.M.，ed.，Oxford University Press，New York，1988；Biocomputing.Informati cs and Genome Projects，Smith，D.W.，ed.，Academic Press，New York，1993；Computer Analysis of Sequence Data，Part1，Griffin，A.M.，and Griffin，H.G.，eds.，Humana Press，New Jersey，1994；Sequence Analysi s in  Molecular Biology，von Heinje，G.，Academic Press，1987；and Seq uence Analysis Primer，Gribskov，M.and Devereux，J.，eds.，M Stockton Press，New York，1991)。
在一些实施方案中，本发明的方法包括将多个线性核酸分子连在一起形成环状核酸分子，例如环状质粒。其中每个目标核酸分子与在所产生的环状产物中形成邻近物的目标核酸分子共享一段同源区域，并按照本发明的方法进行线线重组。目标核酸分子个数是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个或更多个。
在一些实施方案中，至少一个目标线性核酸分子包含选择标志物以允许选择正确的重组体。本发明中可以使用任意合适的选择标志物。在一些实施方案中，选择标志物是抗生素抗性基因，例如，氯霉素，氨苄青霉素，卡那霉素，或杀稻瘟菌素的抗性基因。
所述目标线性核酸分子可以源自任意合适的来源。例如，可以包括来自真核生物或原核生物的核酸序列。在一些实施方案中，所述第一目标线性核酸分子是基因组DNA。典型的，所述基因组DNA是基因组DNA的片段。所述基因组DNA优选包括感兴趣的序列。在一些实施方案中，基因组DNA片段可以通过剪切或消化基因组DNA(例如使用限制性内切酶)获得包含感兴趣的完整序列。在一些实施方案中，所述第一目标线性核酸分子(例如，基因组DNA片段、cDNA文库成员或源自BAC的片段)包括2kb或更长(例如，2.5kb或更长、4kb或更长、5kb或更长、7.5kb或更长、10kb或更长、15kb或更长、20kb或更长、25kb或更长、40kb或更长、50kb或更长、75kb或更长或100kb或更长)的感兴趣的序列。优选的，感兴趣的序列是在第一目标线性核酸分子的任一端的同源臂之间的全部区域。例如，编码次级代谢产物途径或脂肪酸合成途径的基因簇。在一些实施方案中，本发明方法可用于从人类或者非人类动物基因组中直接克隆DNA区域，例如，用于健康研究或通过基因打靶进行修正的再生治疗。例如，在一些实施方案中，所述第一目标核酸分子包括或由来自人或非人类动物的基因组DNA片段组成。所述基因组DNA片段可以包括感兴趣的序列，如包含突变的基因，其中该突变导致疾病或者病症并且将该突变修正为野生型序列可以治疗或预防该疾病或病症。在所述第一目标 核酸分子是基因组DNA片段的实施方案中，所述第二目标核酸分子优选线性化的克隆载体。
在所述第一目标核酸分子是基因组DNA片段的实施方案中，所述方法包括通过消化或剪切基因组DNA来生成第一目标核酸分子以获得包含感兴趣的序列的线性基因组DNA片段，接着使用第一核酸外切酶处理基因组DNA片段与线性克隆载体的混合物，处理包括酶切目标核酸分子和退火使酶切后的目标核酸分子连接的步骤，然后将处理后的核酸分子的混合物共电转化至宿主细胞中。所述第二目标核酸分子优选包含选择标志物。
在一个实施方案中，本公开的方法包括体外连接DNA分子的步骤。
在一个优选实施方案中，所述体外连接步骤包括核酸外切酶消化随后是退火。
在另一个优选实施方案中，所述核酸外切酶是T4聚合酶。
在另一个优选实施方案中，所述体外连接步骤包括Gibson组装。
在另一个优选实施方案中，所述体外连接步骤包括通过DNA聚合酶的DNA合成，所述DNA合成使用或不使用核酸外切酶，随后是退火。
在另一个优选实施方案中，所述体外连接步骤包括由单链退火蛋白促进的退火，所述单链退火蛋白诸如RecA/RAD51、Redβ、RecT、Pluβ或RAD52。
在另一个优选实施方案中，用于同源重组的宿主细胞是大肠杆菌细胞。
在另一个优选实施方案中，用于同源重组的宿主细胞是表达全长RecE和/或ReeT的大肠杆菌细胞。
在另一个优选实施方案中，用于同源重组的宿主细胞是表达全长RecE、RecT和/或Redγ的大肠杆菌细胞。
在另一个优选实施方案中，用于同源重组的宿主细胞是表达截短的RecE、RecT和/或Redγ的大肠杆菌细胞。
在另一个优选实施方案中，用于同源重组的宿主细胞是表达全长RecE和/或RecT的任何细菌宿主细胞。
在另一个优选实施方案中，用于同源重组的宿主细胞是表达Redα、Redβ和/或Redγ的大肠杆菌细胞。
在另一个优选实施方案中，用于同源重组的宿主细胞是酿酒酵母细胞。
提供了用于本发明的试剂盒。在一些实施方案中，所述试剂盒包括编码本文描述的核酸外切酶的核酸。在一些实施方案中，所述试剂盒包括在此处描述的核酸外切酶。优选的，所述第一核酸外切酶是T4DNA聚合酶(T4 DNA polymerase；T4pol)、DNA聚合酶I的Klenow片段(Klenow fragment of DNA Polymerase I；Kle)、T7DNA聚合酶(T7DNA polymerase；T7pol)、核酸外切酶III(Exonuclease III；ExoIII)、Phusion DNA聚合酶(Phusion DNA polymerase；Phu)、T5核酸外切酶(T5 exonuclease；T5exo)、T7核酸外切酶(T7 exonuclease；T7exo)和Lambda核酸外切酶(Lambda exonuclease；λexo)，所述第二核酸外切酶是全长RecE。更优选的，所述试剂盒包括本文描述的宿主细胞。例如，在一些实施方案中，试剂盒中的宿主细胞包括在可诱导启动子控制下编码本文所述的全长RecE、RecT、Redγ和RecA的核酸。所述试剂盒还可包括一个或多个预先制备的线性克隆载体。
本发明的另一优选应用涉及合成生物学中的线性核酸分子的组装，所述线性核酸分子优选线性DNA。因此，在一些实施方案中，所述第一和第二目标核酸分子是线性的，并且所述方法进一步包括在5’到3’核酸外切酶和退火蛋白的存在下将所述第一和第二目标核酸分子与一个或多个其它线性目标核酸分子(例如，1、2、3、4、5、6、7、8、9、至少10、至少20个其它目标核酸分子)接触以产生线性或环状产物。在优选的实施方案中，第一和第二目标核酸分子以及一个或多个其它目标核酸分子之间的同源重组导致基因、操纵子、染色体或整个基因组的产生。DNA核酸的合成生物学组装已经用来产生基因、操纵子、染色体，或在最近用来产生整个基因组。在本发明实施方案中，基于第一核酸外切酶和第二核酸外切酶的联合作用使线性核酸分子组装效率显著提高，本发明将成为商业上和研究中合成生物学DNA组装的一种优选方法。
本发明的另一优选应用是构建单倍型同基因打靶载体，利用本发明的方法从哺乳动物基因组上直接克隆5至10kb的DNA片段作为同基因同源臂，这些DNA片段不仅是相同的基因而且维持了多态性的单倍型，所以发明人称之为单倍型同基因打靶载体，即“HIT”(haplotypic  isogenic targeting)载体。然后通过重组工程将筛选标记以及其它功能元件插入到HIT载体上获得用于打靶的载体。本发明的另一优选应用是对哺乳动物细胞进行基因分型。应用本发明的方法从可能的打靶胚胎干细胞的基因组上克隆含有完整打靶元件的DNA片段，对克隆获得的重组质粒进行限制性酶切分析和DNA测序，根据结果判断细胞是否打靶成功。
实施例
材料和方法
菌株和质粒
E.coli GB2005在DH10B基础上敲除了fhuA，ybcC和recET3，16，44.GB05-dir是在GB2005的染色体上整合了PBAD-ETgA操纵子(PBAD启动子调控下的recE，recT，redγ和recA)3.GB08-red是在GB2005的染色体上整合了PBAD-gbaA操纵子(PBAD启动子调控下的redγ，redβ，redα和recA)16.pSC101-BAD-ETgA-tet 3带有四环素抗性基因和PBAD-ETgA操纵子的温度敏感型质粒，它可以在30℃复制，但是却不能在37℃复制，因此在无选择压力下通过改变温度就能轻易将其消除。
基因组DNA的制备和酶切
革兰阴性细菌明亮发光杆菌(Photobacterium phosphoreum)ANT-2200和发光光杆状菌(Photorhabdus luminescens)DSM15139在50ml培养基中培养过夜。离心收集的细胞悬浮在8ml 10mM Tris-HCl(pH 8.0)中，然后加500ul 20mg ml-1蛋白酶K和1ml 10％SDS，50度温浴2h直至溶液变澄清。酚氯仿抽提和乙醇沉淀获得基因组DNA，最后溶于10mM Tris-HCl(pH 8.0)中。
革兰阳性细菌白色链霉菌(Streptomyces albus)DSM41398在50ml TSB培养基中30度培养2天。根据参考文献45的方法提取基因组DNA，主要流程如下：菌体重悬于8ml SET(75mM NaCl，25mM EDTA，20mM Tris，pH 8.0)中，添加10mg溶菌酶，37度温浴1h，然后加500ul20mg ml-1蛋白酶K和1ml 10％SDS，50度温浴2h直至溶液变澄清，然后加3.5ml 5M NaCl。酚氯仿抽提和乙醇沉淀获得基因组DNA，最后溶于10mM Tris-HCl(pH 8.0)中。
老鼠黑色素瘤细胞B16、人胚胎肾细胞293T和人血的基因组使用 Qiagen Blood&Cell Culture DNA试剂盒提取，步骤略加改动，裂解细胞用蛋白酶K处理后用酚氯仿抽提和乙醇沉淀获得基因组DNA，最后溶于10mM Tris-HCl(pH 8.0)中。
明亮发光杆菌ANT-2200的基因组DNA用BamHI+KpnI酶切以克隆lux基因簇。P.luminescens DSM15139的基因组DNA用XbaI酶切以克隆plu3535-plu3532；用XbaI+XmaI酶切以克隆plu2670。白色链霉菌S.albus的基因组DNA用EcoRV或Cas9-gRNA酶切以克隆盐霉素基因簇。老鼠细胞B 16的基因组DNA用HpaI酶切以克隆Prkar1a，用BamHI+KpnI酶切以克隆Dpy30，用SwaI酶切以克隆Wnt4或Lmbr1l-Tuba1a。人的基因组DNA用NdeI+BstZ17I酶切以克隆IGFLR1-LIN37，用SpeI酶切以克隆DPY30，用BstZ17I酶切以克隆IGFLR1-ARHGAP33，用NdeI酶切以克隆ZBTB32-LIN37。酶切的基因组用酚氯仿抽提和乙醇沉淀，最后溶于ddH2O，浓度大约1ug/ul。
白色链霉菌S.albus基因组DNA的Cas9酶切
S.pyogenes Cas9蛋白购自New England Biolab公司。白色链霉菌S.albus基因组DNA的Cas9酶切在含有80ul 10×Cas9反应缓冲液(NEB)(NEB)，80μg基因组DNA，40μg gRNA-2，40μg gRNA-7和20μg Cas9的800ul反应体系中进行。由于Cas9的切割效率受到DNA底物纯度的严重影响，所以在此试验中，白色链霉菌S.albus基因组DNA需要利用酚-氯仿-异戊醇(25∶24∶1，pH8.0)抽提三次，以保证Cas9切割的效率。37℃温育6小时后，加入100μg RNase A(Thermo Scientific)，37℃温育1小时后加入100μg蛋白酶K(Roche)，继续50℃温育1h。然后将基因组DNA再用酚-氯仿-异戊醇(25∶24∶1，pH8.0)抽提一次，经乙醇沉淀后，溶于适量ddH2O中，使其终浓度在1μg ul-1左右。最后将10μg Cas9切割后的基因组DNA用于本发明方法的克隆实验。
线性克隆载体的制备
PCR扩增(图4)p15A-em载体的模板质粒为p15A-Pamp-luxABECD(购自Genebridge公司)，使用PrimeSTAR Max DNA聚合酶(Takara)，所用引物(表1)含有80个核苷酸的同源臂并通过PAGE纯化。PCR产物通过胶回收来排除引物对后续实验的干扰，所用试剂盒为QIAquick凝胶提取试剂盒(Qiagen)，最后DNA用ddH2O 洗脱，浓度大约200ng/ul，使用200ng进行ExoCET克隆实验。
用来克隆106kb盐霉素基因簇的pBeloBAC11载体和克隆plu3535-3532的pBAC2015载体的构建方法如文献43和文献46所述。通过BamHI酶切暴露出两端的同源臂，然后通过酚-氯仿-异戊醇(25∶24∶1，pH8.0)抽提和异丙醇沉淀收集DNA，最后溶于ddH2O，浓度大约1ug/ul。使用1μg BAC载体进行ExoCET克隆实验。
表1 p15A-cm线性载体扩增的寡核苷酸
Figure PCTCN2017000483-appb-000001
小写字母为同源臂序列。
多片段组装的PCR产物制备
以P.luminescens DSM15139基因组DNA为模板，用PrimeSTAR Max DNA聚合酶(Takara)扩增的PCR产物相互之间带有40bp的同源臂。PCR产物用胶回收纯化，所用试剂盒为QIAquick凝胶提取试剂盒(Qiagen)，最后用ddH2O洗脱，浓度大约200ng/ul。每个片段使用250ng进行组装实验。
mVenus-PGK-neo DNA元件的制备
以pR6K-2Ty1-2PreS-mVenus-Biotin-PGK-em7-neo17为模板，利用PrimeSTAR Max DNA聚合酶(Takara)PCR扩增mVenus-PGK-neo元件。所用引物如表S7所示。PCR产物经试剂盒QIAquick PCR纯化试剂盒(Qiagen)纯化后，用ddH2O，浓度大约为100ng ul-1.使用200ng进行同源重组实验。
体外组装
在0.2ml PCR管中加入10ugDNA分子，200ng 2.2kb p15A-cm线性载体(或者1ug 8kb pBeloBAC11载体)，2ul 10×NE缓冲液2.1和0.13ul 3U ul-1T4 DNA聚合酶(T4pol)，用水补至20ul。然后再PCR仪中进行如下循环：25度1h，75度20min，50度30min，最后4度保温。在多片段组装实验中，25度反应时间为20分钟。反应产物在转化之前用Millipore膜过滤器(Merck-Millipore，cat.no.VSWP01300)脱盐，室温30min。
其他核酸外切酶的反应程序如下：T5核酸外切酶(T5 exo)：50度30min，4度保温；T7核酸外切酶(T7 exo)：25度20min，50度30min，最后4度保温；Klenow大片段(kle)、T7DNA聚合酶(T7pol)、Lambda核酸外切酶(Lam exo)：25度20min，75度20min，50度30min，最后4度保温；核酸外切酶III(Exo III)：37度20min，75度20min，50度30min，最后4度保温；Phusion DNA聚合酶(Phu)：37度20min，50度30min，最后4度保温；Gibson组装(NEB，cat.E2611)的反应程序如下：50度30min，最后4度保温。
电转化感受态大肠杆菌细胞的制备
含有质粒pSC101-BAD-ETgA-tet的E.coli GB05-dir在加有4ug/ml四环素的LB中30度培养过夜(OD600＝3～4)。将40ul过夜培养物(OD600＝3～4)转接到加有合适抗生素的1.4ml LB中，置于Eppendorf热混合器上30度，950rpm培养2h(OD600＝0.35～0.4)。加入35ul 10％ L-arabinose(w/v，in ddH2O)来诱导重组酶(ETgA或gbaA)表达，37度继续培养40min(OD600＝0.7～0.8)。离心收集细胞，9,400g 30sec，2℃。弃上清，沉淀用1ml冰ddH2O悬浮。离心收集细胞，9,400g 30sec，2℃。弃上清，沉淀用1ml冰ddH2O悬浮。重复离心、重悬、再离心，用20ul冰ddH2O悬浮细胞。加入5ul脱盐的体外组装产物，而在mVenus-PGK-neo元件插入实验中则加入200ng质粒和200ng PCR产物的混合物。将细胞和DNA的混合液转入1mm电激杯中，用Eppendorf电穿孔仪2510进行电击，电压1350V，电容10uF，电阻600Ω。加1ml LB至电激杯中，洗涤细胞并将其转移至扎孔的1.5ml管中，置于Eppendorf热混合器上37度，950rpm培养1h。最后将适量的菌液涂布到加有合适抗生素(15ug/ml的氯霉素或15ug/ml的卡那霉素)的LB平板上，37度过夜培养。
实施例1 体外组装和全长RecE/RecT的共同作用提高了直接克隆的效率
发明人以明亮发光杆菌ANT-220047中14kb lux基因簇的直接克隆(图1a)作为模型实验测试了一系列核酸外切酶以及退火方法。在这个克隆实验中，发明人将10μg经过BamHI+KpnI酶切的ANT-2200基因组DNA和200ng 2.2kb的p15A-cm线性载体混合，其中线性载体两端带有与限制性酶切产生的含有lux基因簇的基因组片段两端一致的序列(同源臂)，线性载体通过两端的同源臂与目的基因组DNA片段发生同源重组，形成最终的环状质粒。首先用不同的核酸外切酶在体外处理克隆载体和BamHI+KpnI酶切的基因组DNA，然后将体外反应产物转化到表达了RecE/RecT重组酶的E.coli细胞内。测试的核酸外切酶包括：T4 DNA聚合酶(T4 DNA polymerase；T4pol)，DNA聚合酶I的Klenow片段(Klenow fragment of DNA Polymerase I；Kle)，T7 DNA聚合酶(T7DNA polymerase；T7pol)，核酸外切酶III(Exonuclease III；ExoIII)；Phusion DNA聚合酶(Phusion DNA polymerase；Phu)，T5核酸外切酶(T5 exonuclease；T5exo)；T7核酸外切酶(T7 exonuclease；T7exo)和Lambda核酸外切酶(Lambda exonuclease；λexo)(图2a)。结果显示T4pol，Kle，T5exo和T7exo都能显著提高直接克隆的效率(图2a-e)。核酸外切酶消化之后的退火速率对克隆效率几乎没有影响，发明人选用Eppendorf MC nexus PCR 仪的默认降温速率(2℃ s-1)(图3)。经过测试T4pol的浓度、处理温度和时间对直接克隆效率的影响(图1c，d和图2f)。
发明人分别比较了单独使用全长RecE/RecT、单独使用T4pol体外退火以及联合使用T4pol体外退火和全长RecE/RecT这三种情况下的直接克隆效率(图1f)。在单独使用RecE/RecT时，也能以很高的正确率将14kb的lux基因簇从发光细菌染色体中直接克隆p15A载体上(427)。但是将T4pol体外组装的反应产物转化到一个标准的E.coli工程菌中得到的直接克隆效率要高的多(427vs 4，880)。这表明：(1)E.coli内源的DNA修复体系能够娴熟的将质粒骨架封闭起来；(2)T4pol体外组装的效率很高(4880)；(3)将T4pol体外组装和RecE/RecT细胞内重组结合起来的ExoCET技术却比其中任何一种技术单独作用时都要高效(32500)。
实施例2 同源臂对克隆效率的影响
同源臂越长，克隆效率越高(图1b)。在图5a中，发明人将克隆载体一端的80bp同源臂置于基因簇内部1kb，或者两端均位于基因簇内部1kb，然后对ExoCET的效率与T4pol和RecET的效率进行了比较(图5b)。当载体的两个同源臂均位于最末端时，发明人得到了与之前一样的结果。但是，当一个同源臂或者两个同源臂位于基因簇内部1kb时，只用T4pol进行处理的克隆效率是很低的，这表明T4pol核酸外切酶处理后的退火依赖于末端的DNA序列互补配对。值得注意的是，RecET重组的效率与同源臂的位置几乎没有关系。当一个同源臂位于末端，而另一个同源臂位于内部时得到的实验结果令人深思。当载体和基因组DNA片段之间只有一个末端同源臂时，ExoCET的效率是RecET的12倍，这表明在电转化和RecET重组之前，T4pol体外处理能使两个DNA分子通过一端退火而高效的结合在一起。以上数据表明T4pol只能作用于末端同源臂，而内部同源臂的重组需要RecET的作用。因此，发明人得出的结论是T4pol对ExoCET的主要贡献是通过两个线性DNA分子一端的退火而显著提高它们的共转化效率。然后，再用RecET来促进另一端的重组。在14kb lux基因簇的克隆实验中，当两个同源臂均位于目的基因组酶切片段最末端时，ExoCET的克隆效率是T4pol的6～8倍(图1f和5b)。这说明大部分体外组装产物只有一端结合在了一起(＞85％)，而且体外组装的环状产物和两个线性DNA 分子共转化后完全由RecET产生的重组质粒对ExoCET高效率的贡献很小。在设计ExoCET直接克隆实验时，同源臂位置对克隆效率非常重要。为了获得最高的克隆效率，两个同源臂都要置于目的DNA片段的最末端。实际上，为了发挥体外T4pol核酸外切酶和退火的作用，至少一个同源臂要置于末端。但是另一个同源臂可以置于目的DNA片段的内部，因为RecE/RecT能够对其定位并发生重组。这非常有利于利用直接克隆来构建表达载体，因为其中一个同源臂可以置于目的片段3’端的最末端，而5’端的目的基因可以利用内部同源臂直接置于启动子和核糖体结合位点的控制之下。
实施例3 RecE和ReeT都是ExoCET必需的
因为RecT是一个单链DNA退火蛋白，所以发明人猜测RecT有可能会跟T4pol(3’核酸外切酶)产生的单链DNA区域退火，因此在ExoCET技术体系可能不需要RecE的参与。为了验证这个猜想，发明人将T4pol处理过的DNA底物转化到表达ReeT和Redγ(pSC101-Tg)而且不表达RecE的大肠杆菌细胞内，并没有发现RecT和T4pol之间发生相互作用，因此RecE和RecT都是ExoCET必需的(图5c)。RecA对直接克隆效率有一定的提高。
实施例4 DNA大片段直接克隆的验证
为了验证ExoCET技术的优越性，发明人利用它来做了一些之前用RecET技术很难做的实验。发光光杆状菌(Photorhabdus luminescens)基因组上存在两个大的基因簇：37.5kb的plu3535-3532和52.6kb的plu2670，之前发明人用RecET技术对这两个基因簇进行直接克隆的时候非常困难，其效率分别只有2/12和0/483，而利用ExoCET技术发明人却分别获得了10/12和11/17的正确率(表2)。
表2 利用ExoCET从细菌、哺乳动物细胞和人血中直接克隆的基因组DNA大片段。
Figure PCTCN2017000483-appb-000002
再有就是发明人之前尝试从白色链霉菌(Streptomyces albus)基因组上一步直接克隆106kb的盐霉素基因簇没有成功，因此发明人不得不将其分为三个片段分步克隆，然后再将其拼接成一个完整的基因簇43。但是通过ExoCET，利用一个带有同源臂的BAC载体和EcoRV酶切的基因组DNA，发明人可以直接将106kb的盐霉素基因簇克隆到该BAC载体上，而且获得了2/24的正确率(表2和图6)。因为在106kb盐霉素基因簇的两侧各有一个EcoRV酶切位点，所以发明人可以将其从染色体上释放出来并克隆到载体上。在DNA大片段克隆的时候，有 时候在目的DNA片段的两侧很难找到合适的限制性酶切位点，但是利用可编程的核酸酶则可以摆脱对限制性酶切位点的限制，尤其是RNA介导的核酸内切酶--Cas948，49。为了检测该想法，发明人利用Cas9在很靠近EcoRV的位置将106kb的盐霉素基因簇从染色体上释放出来，然后利用同一个BAC载体上将相同的106kb DNA片段克隆到BAC载体上，最终获得相近的克隆效率(表2和图6)。因此，与RecET相比，ExoCET在DNA大片段的直接克隆上具有显著优越的表现。
接下来发明人测试了ExoCET的效率能否达到从哺乳动物基因组上直接克隆DNA大片段的要求。发明人利用SwaI从老鼠基因组上释放了一个45kb的含有Wnt4基因的片段(图7a)，通过ExoCET发明人获得了8/25的正确率(图7b)。发明人同时也测试了利用Gibson组装1来克隆这一DNA片段。Gibson组装是在体外利用T5exo，Phusion DNA聚合酶和Taq DNA连接酶将相互之间带有同源臂的DNA分子组装起来。通过将Gibson组装的DNA产物转化到阿拉伯糖诱导和不诱导的含有pSC101-BAD-ETgA-tet的E.coli GB05-dir中，发明人获得了大量菌落(181,000和257,000)，而且检测了60个菌落也没有得到一个正确的克隆(图7b)，而且全是自身环化的p15A空载体的。
实施例5 利用ExoCET组装DNA片段
Gibson是为多片段DNA组装建立的方法，所以发明人通过一些DNA多片段组装实验(图7c)对ExoCET和Gibson进行了比较。这些DNA片段都是通过PCR扩增的，而且末端带有40bp的同源臂。在7片段和10片段组装实验中，ExoCET和Gibson组装的效率都是不错的。当Gibson体外组装的产物转化到表达RecE、RecT、Redγ和RecA的大肠杆菌细胞中时(Gibson+ETgA)，组装的效率和正确率均得到了显著提高(图7d)。ExoCET不能组装13个以上的DNA片段，Gibson不能组装16个以上的DNA片段，而Gibson+ETgA却能够将至少20个DNA片段组装成一个54.9kb的质粒。因此，将体外组装和RecET重组结合起来，对于DNA组装的优势是很明显的。
实施例6 利用ExoCET构建单倍型同基因打靶载体
ExoCET还可以用于从包括血液，疾病相关细胞系等来源的哺乳动物基因组上直接克隆DNA片段以助于SNPs的单倍型研究以及为核酸酶介导的人类干细胞打靶来快速构建单倍型同基因(HIT)打靶载体。 从病人、脐带血或体细胞重编程分离到的人类干细胞在生物医学研究的重要性越来越受到人们的重视，对干细胞基因组的精确修饰方法的研究也受到人们的广泛关注。改造人类基因组比改造实验鼠的基因组要更有挑战性，因为人类的遗传多样性很复杂。同基因性(序列相似性)对同源重组的重要性在很多年前人们对老鼠胚胎干细胞进行基因打靶的时候就意识到了14。但是，序列错配对同源重组的影响还未研究清楚，比如单个错配(一个SNP或一个indel)到底能降低多少重组效率？错配距离重组位点的远近是如何影响重组效率的？多个错配是如何影响重组效率的？这些问题目前都还没有搞清楚。无论如何，在基因打靶中使用完全相同的序列很明显是大家极力推荐的，因此ExoCET是一种快速获得理想同源臂的有效方式。与通过PCR从基因组上扩增同源臂的的方法不同，ExoCET不受片段大小的限制，不会引入突变，而且能够维持DNA的单倍型。此外同源臂的末端还可以根据基因分型的方式(比如Southern杂交或者连接PCR)进行选择，所以可以对同源臂的长度进行优化。因此ExoCET为个体化基因组手术提供了优势，尤其是当其与CRISPR/Cas915联合起来时。
发明人利用ExoCET来构建同基因打靶载体来对哺乳动物基因组进行改造。鉴于在老鼠胚胎干细胞研究中的经验50，发明人旨在从人或老鼠基因组上直接克隆5至10kb的DNA片段作为同基因同源臂(图8a和图9a)。值得关注的是这些DNA片段不仅是相同的基因而且维持了多态性的单倍型，所以发明人称之为“HIT”(haplotypic isogenic targeting)载体。通过ExoCET发明人从人(体外培养的细胞系和人血)和老鼠(体外培养的细胞系)基因组中直接克隆了8～9kb的DNA片段(图8b和图9b)，然后通过Redαβ重组工程将筛选标记以及其它功能元件16插入到HIT载体上(图8c和图9c)。
实施例7 利用ExoCET对哺乳动物细胞进行基因分型
ExoCET还可以作为一种对修饰后的基因组进行基因分型的最可靠方法，而Southern杂交和连接PCR会产生假阳性信号。由于在哺乳动物基因分型研究中，长片段(Long Range)PCR很容易产生假阳性信号，所以发明人之前想通过Southern杂交对通过长片段PCR筛选到的Kmt2d-AID-neo打靶的老鼠胚胎干细胞进行确认。但是，发明人始终得不到好的探针。因此，发明人就用图10a所示的ExoCET方法， 从其中4个可能的Kmt2d-AID-neo打靶老鼠胚胎干细胞的基因组上克隆含有完整打靶元件的DNA片段。对ExoCET克隆获得的重组质粒进行限制性酶切分析和DNA测序，结果表明这4个细胞都打靶成功了，而且是单打靶(图10b)。此外，发明人还成功利用ExoCET对之前研究获得的Oct4-Venus-neo，Nanog-Cherry-neo，Gata2-Venus-neo和Set1b-TC-neo打靶老鼠胚胎干细胞进行了再次验证(表3)，这些打靶细胞在之前都是已经通过Southern杂交验证过的。这些结果表明，ExoCET基因分型没有位点限制。发明人之前对一个Klf4-Venus-neo打靶的老鼠胚胎干细胞一直无法确定其是否打靶成功，因为长片段PCR和Southern杂交一直得不到确切的信号。利用ExoCET克隆，发明人在基因组上的相应区域并没有克隆到具有卡那霉素抗性的DNA片段(表3)。对克隆到的氯霉素抗性质粒进行限制性酶切分析发现，其中50％带有野生型的DNA序列(图11)。因此，发明人才确切的知道此细胞并没有被正确打靶。
表3 ExoCET基因分型实验数据
Figure PCTCN2017000483-appb-000003
*经过限制性酶切分析。
**其余10个是分子内重组(在克隆目的序列中含有11个＞40bp的直接重复序列)。
表4 优化ExoCET基因分型中需要的Oct4-Venus-neo打靶的老鼠胚胎干细胞基因组DNA的量
Figure PCTCN2017000483-appb-000004
*经过限制性酶切分析。
与长片段PCR相比，ExoCET基因分型不会产生假阳性信号。而与Southern杂交相比，其操作更简单，不需要繁琐的筛选杂交探针过程。在ExoCET基因分型中，用于释放含有完整打靶元件的限制性酶切位点很容易就能找到，而且在基因组制备好的情况下，只要三天就得到基因分型的结果。更重要的是，ExoCET从来不会产生假阳性信号。由于打靶元件上具有筛选标记，只要500ng限制性酶切的基因组DNA就足以获得较好的克隆效率(表4)。为了提高ExoCET基因分型的通量，可以使用96孔板培养的细胞。
实施例8 ExoCET克隆技术应用于宏基因组样品
对海量基因组测序结果进行功能分析需要简单快速的表达载体构建方法。根据本发明的方法，发明人可以将长达～50kb的DNA片段从3.0x109bp的基因组中克隆出来。为此，发明人将1ng的明亮发光杆菌基因组DNA稀释到10μg芽胞杆菌Bacillus subtilis基因组DNA中来模仿宏基因组，通过ExoCET成功将14kb的lux基因簇成功克隆了出来，并获得了可观的效率(表5)。环境样品通常含有104以上的物种51-53，所以该结果可以激励发明人将ExoCET克隆技术应用于宏基因组样品中。
表5 利用ExoCET从稀释的发光杆菌明亮发光杆菌基因组上直接克隆14kb的lux基因簇。
Figure PCTCN2017000483-appb-000005
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Claims (35)

  1. 一种同源重组的方法，所述方法包括使用第一核酸外切酶处理两个或两个以上目标核酸分子，然后使处理后的目标核酸分子在第二核酸外切酶和退火蛋白的存在下进行同源重组，其中发生同源重组的目标核酸分子之间共享至少一段序列同源区域。
  2. 一种同源重组的方法，其中所述方法包括使用第一核酸外切酶处理第一核酸分子与第二核酸分子，然后使处理后的第一核酸分子与第二核酸分子在第二核酸外切酶和退火蛋白的存在下进行同源重组，其中第一核酸分子和第二核酸分子之间共享至少一段序列同源区域。
  3. 一种组装核酸分子的方法，所述方法包括使用第一核酸外切酶处理两个或两个以上的核酸分子，然后使处理后的核酸分子混合物在第二核酸外切酶和退火蛋白的存在下进行同源重组，其中每个核酸分子与在所产生的组装产物中形成邻近物的核酸分子共享至少一段序列同源区域。
  4. 一种克隆基因组DNA的方法，其中方法包括使用第一核酸外切酶处理基因组DNA片段混合物和线性克隆载体，然后使处理后的基因组DNA片段混合物中的目标DNA片段和线性克隆载体在第二核酸外切酶和退火蛋白的存在下进行同源重组，其中基因组DNA片段混合物中目标DNA片段与线性克隆载体之间共享至少一段序列同源区域。
  5. 根据权利要求1-4的方法，其中所述序列同源区域可以在目标核酸分子内部或末端，优选至少一个同源区域在目标核酸分子末端，更优选同源区域均在目标核酸分子末端。
  6. 根据权利要求5的方法，其中所述同源区域的长度为至少6、至少10、至少20、至少25、至少30、至少40、至少50、至少60、至少70、至少80个核苷酸，优选25个、40个或80个，最优选80个核苷酸。
  7. 根据权利要求1-4的方法，其中所述第一核酸外切酶可以是5’到3’核酸外切酶或是3’到5’核酸外切酶，优选T4 DNA聚合酶、DNA聚合酶I的Klenow片段、T5核酸外切酶、T7核酸外切酶，最优选T4 DNA聚合酶或T5核酸外切酶。
  8. 根据权利要求1-4的方法，其中所述第一核酸外切酶处理包括 体外连接步骤，所述体外连接步骤使两个或两个以上目标核酸分子或者所述第一核酸分子与所述第二核酸分子连接起来，或者使处理后的线性克隆载体和基因组DNA片段混合物中的目标DNA片段连接起来。
  9. 根据权利要求8的方法，其中所述体外连接步骤包括被所述第一核酸外切酶酶切和退火步骤，其中不同核酸分子的酶切可以是单独进行也可以是同时进行，诸如在单个样品中的混合物中。
  10. 根据权利要求8或9的方法，其中所述第一核酸外切酶处理还包括加入DNA聚合酶，dNTP和DNA连接酶的步骤。
  11. 根据权利要求8或9的方法，其中所述第一核酸外切酶处理还包括加入具有3′-5′核酸外切酶活性的DNA聚合酶。
  12. 根据权利要求11的方法，其中所述第一核酸外切酶处理排除添加dNTPs。
  13. 根据权利要求8-10任一项的方法，其中所述第一核酸外切酶处理是T4 DNA聚合酶处理或Gibson组装方法。
  14. 根据权利要求1-4的方法，其中所述第二核酸外切酶是RecE，优选地，所述RecE是重组表达的。
  15. 根据权利要求1-4的方法，其中所述单链退火蛋白包括RecA、RAD51、Redβ、RecT、Pluβ或RAD52，优选其中所述退火蛋白是RecT，更优选地，所述RecT是重组表达的。
  16. 根据权利要求1-4的方法，其中所述同源重组在体外或宿主细胞中进行。
  17. 根据权利要求16的方法，其中所述宿主细胞可以是酵母细胞，优选酵母细胞是酿酒酵母细胞；或者是细菌细胞，优选细菌细胞是枯草芽孢杆菌或大肠杆菌。
  18. 根据权利要求17的方法，其中所述宿主细胞表达核酸外切酶，优选第二核酸外切酶、和退火蛋白。
  19. 根据权利要求18的方法，其中所述宿主细胞是表达全长RecE和RecT的大肠杆菌细胞，优选地，所述宿主细胞是表达全长RecE、RecT和Redγ的大肠杆菌细胞，最优选地，所述宿主细胞是表达全长RecE、RecT、Redγ和RecA的大肠杆菌细胞。
  20. 根据权利要求18的方法，其中所述宿主细胞是表达截短的RecE和RecT的大肠杆菌细胞。
  21. 根据权利要求18的方法，其中所述宿主细胞是表达Redα和Redβ的大肠杆菌细胞。
  22. 根据权利要求18的方法，其中所述宿主细胞通过在质粒载体和/或染色体上表达核酸外切酶，优选第二核酸外切酶、和退火蛋白，优选地，通过质粒载体表达，最优选地，通过质粒载体和染色体同时表达。
  23. 根据权利要求1-4的方法，其中所述目标核酸分子或目标DNA片段是线性的，优选选自用核酸内切酶切割的DNA片段、PCR扩增的DNA片段、基因组DNA片段、cDNA文库成员、源自BAC的片段和克隆载体片段。
  24. 根据权利要求23的方法，其中所述核酸内切酶是限制性内切酶或可编程的核酸内切酶。
  25. 根据权利要求1-4任一项的方法，其中所述目标核酸分子或目标DNA片段的个数是2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个或更多个。
  26. 根据权利要求1-4的方法，其中所述目标核酸分子或目标DNA片段具有长度为0.5kb或更长(例如，1kb或更长、2.5kb或更长、4kb或更长、5kb或更长、7.5kb或更长、10kb或更长、15kb或更长、20kb或更长、25kb或更长、40kb或更长、50kb或更长、75kb或更长或100kb或更长)的序列。
  27. 根据权利要求8或9的方法，其中：
    -所述两种或更多种目标核酸分子、第一目标核酸分子和第二目标核酸分子或目标DNA片段包括一种或多种PCR扩增的DNA片段、基因组DNA片段、cDNA文库成员、和/或衍生自BAC的片段；
    -所述第一核酸外切酶具有3′-5′核酸外切酶活性，优选是T4 DNA聚合酶；
    -所述核酸外切酶处理在体外在不存在dNTPs的情况下进行；
    -所述第二核酸外切酶是全长RecE；
    -所述退火蛋白是RecT；和
    -所述同源重组在表达所述全长RecE和RecT的细菌宿主细胞中进行，优选大肠杆菌。
  28. 根据权利要求8或9的方法，其中：
    -所述两种或更多种目标核酸分子包括三种或多种PCR扩增的DNA片段、基因组DNA片段、cDNA文库成员、或衍生自BAC、线性质粒和/或克隆载体的片段，优选三种或更多种线性质粒和/或克隆载体片段；
    -所述第一核酸外切酶处理包括Gibson组装；
    -所述第二核酸外切酶是全长RecE；
    -所述退火蛋白是RecT；和
    -所述同源重组在表达所述全长RecE和RecT的细菌宿主细胞中进行，优选大肠杆菌。
  29. 试剂盒，其包括权利要求1-28的方法中所述的第一核酸外切酶和第二核酸外切酶或编码权利要求1-28的方法中所述第一核酸外切酶和第二核酸外切酶的核酸。
  30. 权利要求29的试剂盒，其中所述第一核酸外切酶是具有3′-5′核酸外切酶活性的DNA聚合酶，诸如T4 DNA聚合酶、DNA聚合酶I的Klenow片段、T5核酸外切酶或T7核酸外切酶，所述第二核酸外切酶是全长RecE。
  31. 权利要求29或30的试剂盒，其中所述试剂盒还包括表达第二核酸外切酶的宿主细胞，优选地所述宿主细胞中包含编码全长RecE、RecT、Redγ和RecA的核酸。
  32. 权利要求29-31任一项的试剂盒，其中所述试剂盒还可包括一个或多个预先制备的线性载体。
  33. 根据权利要求1-28任一项的方法或权利要求29-32任一项的试剂盒在构建打靶载体中的用途。
  34. 根据权利要求1-28任一项的方法或权利要求29-32任一项的试剂盒在基因分型中的用途。
  35. 根据权利要求1-28任一项的方法或权利要求29-32任一项的试剂盒在DNA合成中的用途。
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