WO2020083083A1 - Dna大片段的克隆方法 - Google Patents

Dna大片段的克隆方法 Download PDF

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WO2020083083A1
WO2020083083A1 PCT/CN2019/111498 CN2019111498W WO2020083083A1 WO 2020083083 A1 WO2020083083 A1 WO 2020083083A1 CN 2019111498 W CN2019111498 W CN 2019111498W WO 2020083083 A1 WO2020083083 A1 WO 2020083083A1
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vector
dna
yeast
gene
fragments
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黄菁
杨波
卢娜
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黄菁
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/09—Recombinant DNA-technology
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    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
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Definitions

  • This application relates to the field of genetic engineering technology. Specifically, it relates to a method for cloning a gene fragment of interest, and the method of the present application is particularly suitable for cloning and editing large DNA fragments with a length greater than 100 kb, especially 300 kb or more.
  • the traditional gene library method can be used to clone genomic DNA of a certain length.
  • bacterial artificial chromosome libraries can only clone up to 300kb of foreign DNA
  • yeast artificial chromosome (YAC) libraries can clone genes from 200kb to 1000kb (Dausset, Ougen et al.1992).
  • YAC yeast artificial chromosome
  • the construction of genomic libraries and the selection of positive clones require a lot of work.
  • the existing YAC library is unstable, and chimerism and gene loss will occur during long-term storage (Scott and Vos 2001).
  • TAR yeast transformation-related recombinant cloning
  • TAR cloning technology is mostly used to clone DNA within 300 kb (Kouprina and Larionov 2006) and it is difficult to clone genes over 300 kb.
  • genes above 300 kb can only be separated from genomic DNA (BAC vectors are generally less than 300 kb).
  • BAC vectors are generally less than 300 kb.
  • this method relies on restriction enzymes to isolate DNA.
  • the gene editing technology ZFNs (engineered, zinc-finger, nuclears), TALENs (transcription, activator-like, efficiency, nuclears)) and CRISPR-Cas9 (clustered regularly, interspaced, short, palindromic, repeats, that are recognized) are advantageous to overcome the disadvantages of The cloning of large fragments of genes.
  • the CRISPR / cas9 system the guide RNA (gRNA) can specifically recognize DNA of 20 nucleic acid sequences, and gRNA can recruit Cas9 to cut at the recognition site.
  • the technology can have a very strong site recognition specificity.
  • Gene editing technology can also be combined with bacterial Red / ET recombination technology to clone large fragments of DNA.
  • the DNA fragments cleaved by CRISPR can also be transformed into bacteria expressing lambda phage Red / ET recombination system together with linear vectors. Gupta et al. 2016).
  • BAC vector loading capacity due to the limitations of BAC vector loading capacity and the conversion of large linear DNA into bacteria, it is theoretically difficult to obtain a single recombinant over 200kb.
  • Yeast transformation-related recombination (TAR) technology can clone 20kb to 250kb of foreign DNA in yeast cells (Kouprina and Larionov 2006).
  • CRISPR / cas9 technology can increase the efficiency of TAR cloning but cannot significantly increase the cloning length of large DNA fragments (Lee, Larionov et al. 2015).
  • the copy number of YAC vector in yeast is low, it is difficult to isolate and purify to obtain a sufficient amount of DNA for the next step of cloning and gene editing.
  • some loci or gene families are much longer than 300 kb. There is currently no cloning method for these very large genes.
  • Bacillus subtilis Bacillus subtilis
  • Bacillus subtilis Bacillus subtilis
  • can assemble large pieces of DNA in cells Itaya, Tsuge et al. 2005; Yonemura, Nakada et al. 2007.
  • the cultivation of Bacillus subtilis is unstable and the DNA transformation ability is low. It is also not suitable for the recombination of multi-segment large genes.
  • genomic DNA of eukaryotes contains a large number of simple repeating sequences.
  • Conventional gene synthesis and PCR methods are difficult to clone these eukaryotic DNA containing a large number of repetitive sequences.
  • These DNA sequences have important effects on the regulation of gene expression and genome stability. Therefore, the assembly method of de novo synthesis of large nucleic acids not only has many technical tedious steps, but also is not conducive to the cloning of eukaryotic genes containing simple repeated sequences.
  • This application provides, for the first time, a method for efficiently, quickly and easily cloning DNA fragments above 100 kb, especially above 300 kb.
  • genome editing technologies such as Crispr / Cas9 technology, TAR cloning technology and yeast DNA homologous sequence-dependent recombination assembly technology to quickly achieve large fragments (more than 100kb, especially more than 300kb) Editing and cloning of genomic DNA, especially eukaryotic genomic DNA.
  • YAC vectors with autonomously repeated sequence (ARS) deletions can be used for the cloning of genomic DNA containing ARS-like sequences (Noskov, Koriabine et al. 2001). But this method is selective for the target gene.
  • the negative screening method using URA3 gene activity can be used to clone any eukaryotic gene and prokaryotic gene.
  • the present application can clone genomic DNA containing simple repeated sequences.
  • this application can easily prepare a series of 100kb to 200kb, even greater than 200kb using Crispr / TAR cloning technology DNA fragment with terminal homology sequence.
  • this application can realize the assembly of multiple genes of more than 100 kb into genes of more than 300 kb in yeast. Therefore, the method of this application can not only clone DNA fragments of more than 300 kb, but also greatly reduce the difficulty of cloning , Increasing the success rate.
  • the method of the present application is also faster and more efficient than the methods in the prior art in cloning prokaryotic genes.
  • the efficiency of assembling large pieces of DNA in yeast depends on the number of DNA fragments converted into yeast and the number of moles of DNA fragments.
  • the method of the present application can easily prepare a large amount of DNA (more than 100 kb) for homologous recombination using yeast TAR cloning technology, which greatly reduces the number of DNA fragments used for assembly.
  • the method of the present application utilizes shuttle vectors to amplify the target fragments in large numbers in microorganisms such as bacteria; uses homing endonucleases to completely cut the target large fragments; and uses the negative screening system of URA3 to avoid false positives caused by the self-cyclization of the vector; Since the digested product can be directly used for transformation, DNA loss and DNA fragmentation caused by DNA gel recovery are avoided.
  • the above design ensures that a sufficient amount of complete large DNA fragments are used for the assembly of yeast cells, which greatly increases the efficiency of cloning of large DNA fragments.
  • this application provides, for the first time, a method for efficiently and quickly cloning and editing DNA fragments above 100 kb, especially above 300 kb.
  • the present invention relates to the following aspects:
  • the present invention provides a method for cloning a gene fragment of interest, which includes the following steps:
  • step 5) Transform the multiple DNA fragments with homologous ends obtained in step 4) together with the linearized vector into a microorganism with homologous recombination activity, preferably yeast, so as to assemble in the cell to obtain multiple genes Recombinants of fragments arranged in a certain order.
  • a microorganism with homologous recombination activity preferably yeast
  • the step 1) includes cutting the chromosome or vector using genome editing technology to obtain the gene fragment of interest.
  • the genome editing technology is selected from one or more of the following: zinc finger nucleases (zinc finger nucleases, ZFNs), transcription activator-like effector nuclease technology (transcriptionactivator-like effector nuclears, TALENs) and clustered regularly spaced short palindrome repeats (Clustered Regularly Interspaced Shorted Palindromic Repeat, CRISPR) technologies include CRISPR / Cas9 and CRISPR-Cpf1, preferably CRISPR / Cas9.
  • the step 2) includes using homologous recombination splicing technology to clone the gene fragment of interest into the additional vector, preferably a shuttle vector.
  • the homologous recombination splicing technology is selected from one or more of the following: sequence-dependent in vitro assembly technology (such as Gibson assembly, SLIC, LIC, etc.), transformation-coupled recombination of yeast (TAR ) Technology and bacterial Red / ET homologous recombination technology, preferably yeast transformation coupled recombination (TAR) technology.
  • sequence-dependent in vitro assembly technology such as Gibson assembly, SLIC, LIC, etc.
  • transformation-coupled recombination of yeast (TAR ) Technology and bacterial Red / ET homologous recombination technology preferably yeast transformation coupled recombination (TAR) technology.
  • the DNA fragment obtained in step 4) is a eukaryotic gene fragment, which is a DNA fragment of about 100 kb to 300 kb, and the The DNA fragment has homologous ends, preferably 60-800 bp, more preferably 90-200 bp homologous ends.
  • the linearized vector in the method for cloning a gene fragment of interest provided by the present application, is a linearized vector having a homologous end sequence of the DNA fragment of interest, preferably a linearized yeast shuttle vector.
  • the linearized vector is a YAC or TAR cloning vector.
  • the linearized vector is a YAC cloning vector, preferably a pTARYAC-TRP1 cloning vector.
  • the additional vector is selected from one or more of the following: BAC (bacterial artificial), YAC (Yeast artificial artificial chromosomes), and PAC ( P1artificial chromosomes), preferably a BAC vector.
  • the yeast in the method for cloning a gene fragment of interest provided by the present application, is Saccharomyces cerevisiae.
  • the microorganism used for amplification is a bacterium, preferably E. coli, for example, ElectroMAX TM DH5 ⁇ -E TM Competent cells (Invitrogen, Cata: 11319019) .
  • the gene fragment of interest includes prokaryotic and eukaryotic gene fragments, preferably eukaryotic gene fragments, preferably having a length exceeding 100 kb, preferably exceeding 300 kb, more Preferably it exceeds 400 kb.
  • the restriction enzyme is a homing enzyme, such as I-CeuI, I-SceI, PI-PspI, PI-SceI Wait.
  • the cell in the method for cloning a gene fragment of interest provided by the present application, is a cell having DNA homologous recombination activity, preferably a yeast cell, such as Saccharomyces cerevisiae.
  • the method further comprises screening the recombinant gene containing the gene fragment of interest obtained in step 5) using the URA3 resistance screening system.
  • the screening includes:
  • the method further includes the following steps between steps 4) and 5): repeating steps 1) -4)
  • the DNA fragments have homologous ends, preferably 60-800 bp, more preferably 90-200 bp homologous ends.
  • the method further includes performing one or more steps 4) after step 5) to obtain multiple homologous ends
  • the DNA fragment is preferably 100 kb or more, which is then subjected to step 5) to obtain recombinants of 300 kb or more arranged in a certain order.
  • the present application provides a method for screening a YAC vector recombinant with a foreign gene using the URA3 resistance screening system, which includes the following steps:
  • step 2) clone the recombinant vector obtained in step 1) into yeast cells;
  • step 3 Screen the yeast cells containing the recombinant vector obtained in step 2) on the selection medium containing 5-FOA and the corresponding auxotrophs.
  • Figure 1 Schematic diagram of high-efficiency subgenome cloning technology, showing the gene editing-dependent subgenomic rapid DNA cloning technology.
  • Figure 3 Design and sequence of gRNA on BAC vector.
  • FIG. 4 Schematic diagram of gRNA structure.
  • crRNA and tracrRNA are shown paired.
  • the variable target-specific prototype spacer of crRNA is represented by "N" bases.
  • CRISPR / Cas9 digestion results in vitro. After CRISPR / Cas9 digestion in vitro, the genomic DNA and the vector that did not carry the target DNA were identified by DNA electrophoresis. CRISPR / Cas9 digestion and identification in vitro include the following steps:
  • Step 1 Mix equimolar tracrRNA and CrRNA to form gRNA;
  • Step 2 Mix equimolar gRNA and Cas9 to form RNP complex
  • Step 3 RNP complex extracellular digestion DNA template
  • M represents a DNA marker
  • Lane 1 is Cas9 0ug
  • Lane 2 is Cas 9 0.125ug
  • Lane 3 is Cas 9 0.25ug
  • Lane 4 is Cas 9 0.5ug.
  • FIG. 1 Map of yeast bacterial shuttle plasmid pTARBAC-TRP1 generated with SnapGene TM .
  • FIG. 7A and 7B Schematic diagram of PCR screening of TAR positive clones.
  • Figure 7A is a schematic diagram of primer design.
  • Figure 7B is a PCR positive clone identification diagram.
  • M represents a molecular weight of 100 bp
  • lane 1 represents an empty plasmid PCR product
  • lanes 2-4 represent recombinant positive clones.
  • FIG. 8 TAR and CRISPR / Cas9 combined technology is used to clone the target gene fragment from BAC vector.
  • FIG. 11 Map of yeast bacterial shuttle plasmid pTARBAC-HIS5 generated with SnapGene TM .
  • FIG. 13A and 13B PCR method to identify multi-segment yeast transformants.
  • M represents a molecular weight control
  • lanes 1-4, 7-14 represent empty vector PCR products
  • lanes 5, 6, 15 represent recombinant transformant PCR products.
  • FIG. 13A M represents a molecular weight control
  • lanes 1-4, 7-14 represent empty vector PCR products
  • lanes 5, 6, 15 represent recombinant transformant PCR products.
  • restriction enzyme site refers to a target nucleic acid sequence that is recognized and cleaved by a restriction enzyme. Restriction enzymes are well known in the art.
  • target gene fragment refers to a target DNA fragment that needs to be cloned, and may be a genomic fragment or a synthetic foreign fragment, or a complete gene.
  • the term “genome” includes naturally occurring genomes and synthetic genomes, and includes genetically engineered genomes, such as those that did not previously exist in laboratories and nature, which include modified genomes and contain nucleic acids and / or parts from more than one species Genome hybrid genome.
  • the term “genome” includes organelle genomes (eg, mitochondrial and chloroplast genomes), genomes of self-replicating organisms (cellular genomes), including prokaryotic and eukaryotic organisms, fungi, yeast, bacteria (eg, mycoplasma), archaea, vertebrates , Mammals and other organisms, and viral genomes and other genomes that rely on host proliferation.
  • the genome also includes those organisms and synthetic organisms that do not fall within any known Linnean classification.
  • An exemplary genome may be a microbial genome, such as the genome of a single-celled organism including bacteria and yeast.
  • the prior art lacks a method for cloning large genomic DNA above 100 kb, especially over 300 kb, especially eukaryotic DNA containing complex sequences.
  • eukaryotic genes contain many simple repetitive sequences, which are difficult to synthesize using gene synthesis and PCR extension methods. For genes longer than 100 kb, it is also difficult to find specific restriction enzymes to isolate large fragments of genes.
  • the present invention establishes a method for efficiently cloning genes over 100 kb, especially over 300 kb, especially eukaryotic genes.
  • the method of the present application is not only efficient, omitting de novo synthesis or PCR-dependent gene amplification steps, but can also clone eukaryotic genes containing a large number of simple repeating sequences.
  • the method of the present application can quickly isolate large subcloned gene fragments using gene editing technology and homologous sequence-dependent DNA recombination technology. Secondly, this method can also be used to purify genomic DNA containing simple repeated sequences. Finally, using yeast's homologous recombination mechanism, multiple foreign gene fragments of more than 100 kb are assembled into a single recombinant of up to 2 Mb in yeast.
  • This method can not only be used to clone eukaryotic genomic DNA of more than 100kb, especially more than 300kb, but also can efficiently clone large fragments of prokaryotic genome or DNA virus genome of the same length, not only can be used to clone consecutive gene families on chromosome Or loci, you can also edit loci at different locations on a chromosome.
  • the present application is an efficient genome cloning technique for large-segment (more than 100 kb, especially more than 300 kb) DNA.
  • the technical solution of the present application mainly includes the following aspects: 1. Using genome editing technology to cut specific DNA fragments of chromosomes or vectors; 2. Cloning these fragments using the DNA recombination principle unique to microbial cells' homologous sequence dependence Into a new vector; 3 use the characteristics of the shuttle vector to amplify in microorganisms, and purify DNA fragments from 100 kb to 300 kb from the vector by restriction enzymes, especially homing endonuclease; 4.
  • the series has 60-800bp, preferably 90-200bp homologous end DNA fragments, and these fragments are transformed into Saccharomyces cerevisiae together with linear yeast vector (YAC), and the recombinants with a length of more than 300kb are obtained by utilizing the homologous recombination characteristics of yeast.
  • YAC linear yeast vector
  • the method for cloning a target gene fragment of the present application involves the following steps:
  • Gene editing technologies include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly) interspaced short-palindromic repeats. Intermittent short palindrome repeat sequence) / Cas9 technology. Because their recognition sequences are relatively long, it is easier to specifically cleave longer gene fragments than restriction enzymes. Among the existing gene editing technologies, CRISPR / Cas9 technology is simpler and faster. In addition to the classic CRISPR / Cas9 system, the CRISPR-Cpf1 developed in recent years has also been used to edit genomes rich in AT regions.
  • CRISPR / Cas9 in vitro digestion can refer to the existing technology, and can also be divided into the following steps:
  • RNP complex digests DNA template extracellularly and reacts at 37 °C for 2 hours.
  • the homologous sequence-dependent recombination splicing technique is preferably used to clone the target gene fragment into another gene vector to obtain a sub-recombinant.
  • Sequence-dependent recombination and splicing technologies include sequence-dependent in vitro assembly technology (such as Gibson assembly, SLIC, LIC, etc.), yeast transformation-coupled recombination (TAR) technology and bacterial Red / ET homologous recombination technology, cloned into some gene carriers
  • sequence-dependent in vitro assembly technology such as Gibson assembly, SLIC, LIC, etc.
  • yeast transformation-coupled recombination (TAR) technology and bacterial Red / ET homologous recombination technology, cloned into some gene carriers
  • BAC bacterial artificial
  • YAC Yeast artificial
  • PAC P1artificial
  • the characteristics of gene vectors can be used to transform yeast vectors carrying foreign genes into non-yeast microorganisms, preferably bacteria, so that the obtained subrecombinants can be found in microorganisms, such as bacteria Was amplified.
  • a large amount of vector DNA within 300 kb can be prepared from bacteria. It is known that the size of DNA fragments can affect their conversion efficiency within microorganisms. For example, the efficiency of transforming 240Kb plasmid in bacteria is 30 times lower than that of transforming 80Kb plasmid DNA. Therefore, we selected highly efficient competent strains, such as ElectroMAX TM DH5 ⁇ -E TM Competent cells (Invitrogen, Cata: 11319019) to transform DNA.
  • the additional vector is digested by restriction enzymes designed in the vector to obtain DNA fragments.
  • the DNA fragment is a DNA fragment of about 100 kb to 300 kb, and the DNA fragment has homologous ends, preferably 60-800 bp, more preferably 90-200 bp homologous ends.
  • the restriction enzyme is preferably a homing enzyme.
  • homing endonucleases in the gene cloning method of the present application.
  • Homing endonucleases include I-CeuI, I-SceI, PI-PspI, PI-SceI, etc. They are double-stranded DNA nucleases that can recognize longer non-palindrome sequences (12-40bp).
  • the recognition sequence of homing endonuclease is very rare. For example, the 18 bp recognition sequence appears only once in every 7X10 10 bp equivalent to the total length of 20 mammalian genomic DNA. Therefore, the use of homing endonucleases can separate any foreign gene fragments from the vector.
  • homing endonucleases can be used to remove fragments of any length from the gene vector. For example, the complete enrichment in pTARYAC-TRP1 vector is isolated.
  • the DNA fragment preferably a sequence with a homologous end of 60-800 bp, preferably 90-200 bp, is transformed into yeast together with a yeast vector, preferably a linear yeast vector, and a recombinant containing the gene fragment of interest is obtained using the homologous recombination mechanism of yeast .
  • Yeasts as host cells include Saccharomyces cerevisiae and other yeast species such as Saccharomyces pombe, which can be used to clone ultra-long genomic DNA. Due to their unique set of genetic manipulation tools, yeast hosts are particularly suitable for manipulation of donor genomic material. The natural capabilities of yeast cells and decades of research have produced a rich set of tools for manipulating DNA in yeast. These advantages are well known in the art. For example, yeast, using their rich genetic system, can assemble and reassemble nucleotide sequences by homologous recombination, a capability that many readily available organisms do not have. Yeast cells can be used to clone larger DNA that cannot be cloned into other organisms, such as whole cells, organelles, and viral genomes. Therefore, one embodiment of the described method takes advantage of the great power of yeast genetics to advance synthetic biology and synthetic genomics by using yeast as host cells for manipulating the genomes and synthetic genomes of difficult and other organisms.
  • yeast especially Saccharomyces cerevisiae, has great advantages as a host cell for cloning DNA fragments.
  • yeast has strong recombinase activity, and can connect DNA fragments of up to 25 overlapping fragments to carrier DNA at a time (Gibson, Benders et al. 2008), so it has the ability to ingest multiple DNAs.
  • the homologous recombination ability of yeast can effectively assemble multiple fragments of DNA into a single recombinant.
  • yeast artificial chromosomes contain centromeres that can support the replication of eukaryotic unstable DNA of at least 2Mb (Kouprina, Leem et al. 2003). Large prokaryotic DNA fragments can be cloned in yeast using the universal genetic code. Toxic gene expression is usually not an obstacle to cloning donor nucleic acids in yeast. For example, studies on the genomes of bacteria and archaea indicate that since eukaryotes and these bacteria use different protein expression machinery, proteins expressed from cloned genomes pose little risk of harm to yeast hosts. Transcription signals in yeast are different from transcription and translation in bacteria. In fact, most prokaryotic genes are probably not expressed in yeast. There is no restriction barrier in yeast.
  • a barrier it may be a replication barrier rather than a gene expression barrier. Genotoxicity is minimized because the regulation of gene expression in eukaryotes such as yeast is different from that in prokaryotes. Moreover, Mycoplasma uses the codon UGA as tryptophan rather than as a translation termination signal. Therefore, most of the Mycoplasma genes, if expressed, will produce truncated proteins in yeast. This largely avoids the possibility of toxic gene products.
  • these DNA fragments containing homologous sequence ends and vectors such as gene vectors, including linear YAC vectors, into yeast together.
  • vectors such as gene vectors, including linear YAC vectors
  • yeast's unique high-efficiency DNA homologous recombination mechanism these gene fragments can assemble circular YAC vectors in yeast cells.
  • the vector contains any DNA elements (eg, origin of replication) required to promote vector replication in one or more desired cell types and selection and / or resistance markers for use in different cell types.
  • DNA elements eg, origin of replication
  • Resistance markers are well known. The skilled person can determine suitable resistance markers for different host / donor combinations. In some cases, it is desirable to use non-clinically relevant markers. In other cases, the choice of resistance marker depends on the nature of the donor, host, and / or recipient cells.
  • the yeast is preferably Saccharomyces cerevisiae.
  • Yeasts such as Saccharomyces cerevisiae have an efficient homologous recombination mechanism, as long as there are more than 60bp homologous segments between two DNA molecules, homologous recombination can be carried out accurately and efficiently (Noskov, Koriabine et al. 2001).
  • gene editing technology and yeast TAR cloning technology can be used to generate a series of large gene fragments with homologous ends.
  • the size of these gene fragments is between about 100 kb and 300 kb, and they have homologous ends, and the length is between 60 bp and 800 bp, preferably between 90 bp and 200 bp.
  • the following primers were designed to amplify the YAC shuttle vector pYACTAR-TRP1, which was used to clone part of the human IGL locus sequence in the BAC vector (RP11-890G10).
  • the capital letters of the primer sequence indicate the recognition site of the endonuclease I-SceI.
  • the upstream of the I-SceI recognition site ((60nt), indicated by lowercase letters) is the target gene to be cloned in the BAC vector (RP11-890G10)
  • the homologous sequence, downstream of the I-SceI recognition site ((30nt), indicated by lowercase letters) is the recognition sequence of the amplified DNA of the pYACTAR-TRP1 vector. After amplifying the pYACTAR-TRP1 vector by PCR, the ends of the linear vector contain homologous sequences that require cloning of DNA fragments.
  • Reverse primers used to amplify the YAC shuttle vector pYACTAR-TRP1 sequence 4: SEQ ID NO: 4
  • DNA fragments can be adapted into a single recombinant in yeast cells in a certain order. Therefore, by using the homologous recombination mechanism of yeast, multiple DNA fragments with homologous ends can be homologously recombined, thereby connecting into a single recombiner, thereby realizing the splicing of multiple large fragments of DNA in yeast cells.
  • linear recombinants can also be linked into circular YAC recombinants by linear YAC vectors.
  • This circular YAC vector contains centromeres and can distribute recombinants to parental and progeny cells during replication.
  • the circular structure can prevent the linear foreign DNA from being hydrolyzed by DNase in yeast.
  • the principle of YAC vector expressing Saccharomyces cerevisiae-dependent multi-segment assembly is shown in Figure 10.
  • the self-circulating vector can express URA3 in yeast cells.
  • the enzyme encoded by the URA3 gene prevents yeast cells from growing on medium containing 5-FOA. It has been reported that when the distance between the TATA box of the ADH1 promoter and the transcription initiation point exceeds 130 bp, the ADH1 promoter loses transcriptional activity for downstream genes (Furter-Graves and Hall, 1990). When the foreign gene is recombined between the ADH1 promoter and URA3, the yeast cells do not express URA3, so they are not sensitive to 5-FOA and can grow on the medium containing 5-FOA. This screening method can increase the screening frequency of positive recombinants. The statistical results are shown in Figure 9.
  • ARS autonomously duplicated sequence
  • the human IGL locus is located in the long arm of human chromosome 22 on the 1st, 1st, and 2nd subbands. Due to different human haplotypes, this locus includes 70 to 71 IGLV variable region genes, 7 to 11 IGLJ junction region genes and 7 to 11 IGLC conserved region genes, each of which is followed by a conserved region gene. According to the genome version GRCh37 / hg19, the human IGL locus starts and ends on the chromosome between 22,385,572 and 23,265,082 of human chromosome 22, with a total of 879,511 bases. According to the UCSC genome browser display (https://genome.ucsc.edu/), find some BAC vectors covering the above area.
  • BAC carrier information and start and stop positions are shown in Figure 2. These BACs are purchased from BACPAC Resources Center (BPRC) and are 160kb to 200kb in length. There is at least a few tens of kb overlapping area between each adjacent BAC vector.
  • BAC vector DNA was purified by BAC extraction kit, such as Takara's NucleoBond Xtra BAC (Cata #: 740436.25), and stored in a refrigerator at 4 ° C.
  • genomic target fragments 100 kb to 200 kb
  • CRISPR / Cas9 system for digestion, gRNA has fewer non-specific recognition sites on the BAC vector than the whole genome, ensuring the specificity of cleavage and reducing off-target effects.
  • the overlapping regions of adjacent BAC vectors in the BAC genomic library generally have dozens or dozens of kb.
  • the adjacent BAC plasmids on the genome need to be Design the gRNA in the overlapping area, and use Cas9 enzyme to cut the target DNA from the BAC plasmid.
  • Cas9 enzyme to cut the target DNA from the BAC plasmid.
  • simple reverse repeat regions should be avoided, and the distance between the gRNA target sequences in the overlapping region should not exceed 1 kb, so as to facilitate the design of primers for TAR cloning in this region.
  • the searched feature sequence is [5'-G19nt-NGG].
  • software such as Blat search ( http://genome.ucsc.edu/cgi-bin/hgBlat ) is also needed to analyze the potential off-target sequences of the genome, especially to avoid the off-target sequences appearing in the BAC vector.
  • the design results are shown in Figure 3.
  • gRNA is composed of CRISPR RNA (crRNA) and trans-activating crRNA (trans-activating crRNA, tracrRNA).
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • a part of the sequence of crRNA can be paired with tracrRNA to form a double-stranded RNA structure; a part of the sequence is complementary to the target target to identify the target sequence .
  • TracrRNA not only stabilizes crRNA, but also participates in the binding and cleavage of Cas9 protein and DNA.
  • CrRNA and tracrRNA can be obtained not only by reverse transcription using template DNA containing the T7 promoter, but also by direct chemical synthesis.
  • chemical synthesis of Alt-R CRISPR crRNA and Alt-R CRISPR-Cas9tracrRNA (IDTdna, Cata # 1072533) from IDTdna. The sequence and structure are shown in Figure 4.
  • the CRISPR / Cas9 digestion method in vitro is as follows:
  • RNP Cas9 ribonucleoprotein
  • FIG. 5 shows the results of digestion of BAC plasmid DNA (RP11-890G10) with gRNA / Cas9.
  • gRNA consists of CrRNA and tracrRNA. The sequences of CrRNA are:
  • IGL2A (rGrCrUrCrArCrUrGrGrGrGrCrCrArGrCrArGrCrArGrCrArGrCrUrGrUrUrArGrArGrCrUrGrArGrCrUrGrCrUrGrCrUrGrCrUrGrCrUrGrGrCrUrGrGrCrUrGrGrCrUrGrGrCrUrGrGrCrGrGrGrCrGrGrGrGrCrGrGrGrGrCrUrUrAGrArGrCrUrGrCrUrGrCrUrUrU).
  • tracrRNA was purchased from IDTdna
  • Figure 5 from left to right are 1kb plus DNA (NEB) Cat #: N0552S and under different Cas9 enzyme equivalent effects, BAC (RP11-890G10) was digested by different levels of CRISPR / Cas9 complex.
  • the isolated target gene fragment can be transformed into yeast cells together with the linear yeast bacterial shuttle plasmid (pTARYAC-TRP1).
  • pTARYAC-TRP1 linear yeast bacterial shuttle plasmid
  • yeast cells the gene fragment of interest and linear pTARYAC-TRP1 are assembled into a circular YAC vector by homologous sequences at their respective ends.
  • the shuttle plasmid pTARYAC-TRP1 (as shown in Fig. 6) was transformed from pBACe3.6 (purchased from BACPAC Resources Center, Cata #: pBACe3.6). Part of the DNA comes from the plasmid pBACe3.6 and contains bacterial replication sequences and selectable markers, such as the repE gene, sopA gene, sopB gene, oriT1 replicon, and the selective resistance gene Chloramphenicol resistant gene (CMR); the other part DNA is derived from genetic chemical synthesis and contains yeast-related replication sequences and selection resistance genes, such as replication initiation sequence ARS4, centromere domain CEN5, resistance genes TRP1, URA3, etc. The synthetic gene sequence is shown in Sequence 1, SEQ ID NO: 1. Yeast strain AB1380 can be purchased from the American Type Culture Collection ( 20843). Its gene phenotype is MATa ade2-1lys2-1can1-100trp1 ura3his5.
  • the I-SceI site was introduced between the ADH1 promoter of pTARYAC-TRP1 and the URA3 gene transcription start site.
  • the restriction enzyme I-SceI was used to linearize pTARYAC-TRP1.
  • the primer sequences are as follows:
  • Reverse primers used to amplify the YAC shuttle vector pYACTAR-TRP1 (Sequence 4: SEQ ID NO: 4):
  • the 3 'end of the primer (shown in lower case letters), about 30 bp, is the same as the end of the linearized vector.
  • the 5 'end (shown in lower case) of the primer has more than 60 bases, which is the same as the terminal sequence of the gene fragment to be cloned in the BAC vector.
  • Between the 5 'and 3' ends of the primer (shown in capital letters) is the endonuclease I-SceI recognition site.
  • TaKaRa LA DNA Polymerase TaKaRa RR002A
  • I-SceI I-SceI
  • TAR and CRISPR / Cas9 combined technology for cloning target genes from BAC vector is shown in Figure 8.
  • 100ng of CRISPR / Cas9 linearized target gene and 100ng of PCR linearized vector were co-transformed into competent yeast AB1380.
  • the yeast cells were smeared in a selective solid synthesis containing 5-fluoroorotic acid (5-Fluoroorotic acid hydrate, 5-FOA, Sigma F5013) and TRP And culture on medium (Katara, Cata #: 630309).
  • the basic "conversion mixture” (Sigma Cata # YEAST1-1KT) consists of the following components: 240 ⁇ l PEG (50% w / v), 36 ⁇ l 1.0 mol / L lithium acetate. After mixing 25 ⁇ l of linear vector DNA (2.0 mg / ml) and 50 ⁇ l of Cas9 digested linearized DNA (0.1-10 ⁇ g), each reaction tube was vigorously shaken until the cells were completely mixed, which usually takes about 1 minute. Incubate at 30 ° C for 30 minutes, then heat shock in a 42 ° C water bath for 20-25 minutes. Centrifuge at 6000-8000r / min for 15 seconds, and remove the conversion mixture with a micropipette.
  • Two primers are designed at opposite ends of the linear vector to amplify the empty vector, and the third primer is designed to be upstream of the homologous recombination sequence at the end of the target gene and used to amplify the inserted positive clone. See Figure 7A for primer design.
  • the sequence of the primers is as follows: Primer 1 (sequence 5: SEQ ID NO: 5) for amplifying an empty vector: CATCAGCTCTGGAACAACGA; Primer 2 (sequence 6: SEQ ID NO: 6) for amplifying an empty vector: GGCAACCAAACCCATACATC; Primer 3 (sequence 7: SEQ ID NO: 7) to amplify the inserted positive clone: AAAGGCTCAACAGGTTGGTG.
  • the PCR identification results are shown in Figure 7B.
  • Band 1 is the PCR product of the empty vector.
  • Bands 2, 3, and 4 are PCR products of positive clones.
  • the linear vector pTARYAC-TRP1 easily cyclizes itself in yeast cells, many negative clones are generated, which leads to many false positive results.
  • the URA3 resistance screening system in which 5-FOA was used as a negative screening drug.
  • the self-circulating vector can express URA3 in yeast cells.
  • the enzyme encoded by the URA3 gene prevents yeast cells from growing on medium containing 5-FOA.
  • the negative screening method of URA3 gene activity can significantly increase the screening frequency of positive recombinants, screening out 77% of the false positive results.
  • the negative screening method using URA3 gene activity can be used to clone any eukaryotic gene and prokaryotic gene.
  • the recombinant pTARYAC-TRP1 vector can be amplified in bacteria. This is because the homing endonuclease is designed in the middle of PCR primers, such as I-SceI site, and the recognition sequence of homing endonuclease in the genome is extremely low. Therefore, the homing endonuclease can completely separate the target fragments of any length from the pTARYAC-TRP1 vector.
  • the method of amplifying the target gene from bacteria is as follows:
  • EZNA Yeast DNA Kit Omega Bio-tek, D3370-01
  • 100 ng of DNA was added to 50 ⁇ of competent bacteria ElectroMAX TM DH5 ⁇ -E TM Competent cells (Invitrogen, Cata # 11319019). The electric conversion conditions are 1350V and 5ms pulse.
  • 1 ml of SOC medium (Invitrogen, Cata # 15544034) was added, and cultured at 37 ° C for one hour. 100 ⁇ l of the culture was inoculated into an LB petri dish containing chloramphenicol (15ug / ml) and incubated at 37 ° C overnight.
  • yeast Due to the low copy number of YAC vectors in yeast, yeast grows slower than bacteria, YAC vectors and linear yeast chromosomes are difficult to separate in vitro, and large fragments of DNA are easy to break.
  • the size of circular YAC vectors directly extracted from yeast is not More than 500kbp. Therefore, the characteristics of the shuttle vector pTARYAC can be used to transform genes cloned in yeast into bacteria and amplify the target fragments in bacteria.
  • the efficiency of bacterial transformation will be reduced.
  • the efficiency of transforming 240Kb plasmid in bacteria is 30 times lower than that of transforming 80Kb plasmid DNA.
  • the conversion efficiency is at least 10 times higher than the traditional conversion method, we chose the electric conversion method to convert large molecular weight DNA.
  • the transformation efficiency of ElectroMAX TM DH5 ⁇ -E TM Competent cells is> 1x 10 10 clones / ⁇ g. Because recA1mutation can increase the stability of foreign DNA, it is believed that other highly competent competent bacteria containing recA1mutation are suitable for amplification of plasmids containing large fragments of DNA.
  • homing endonucleases including I-CeuI, I-SceI, PI-PspI, PI-SceI, etc., are double-stranded DNA nucleases that can recognize longer non-palindrome sequences (12-40bp) .
  • the recognition sequence of the homing endonuclease is very rare. For example, the 18bp recognition sequence only appears once every 7X10 10 bp equivalent to the total length of 20 mammalian genomic DNA, so the homing endonucleases designed in the vector can completely separate any foreign gene fragments from the vector .
  • Saccharomyces cerevisiae has an efficient homologous recombination mechanism. As long as there are more than 60bp homologous segments between two DNA molecules, homologous recombination can be performed accurately and efficiently (Noskov, Koriabine et al. 2001).
  • a series of large gene fragments with homologous fragment ends can be generated. These gene fragments can range in size from 30 kb to 300 kb, and they have homologous ends between each other, and are between 90 bp and 200 bp in length. There are many repeated simple sequences in eukaryotic genomic DNA, so avoid designing homologous ends in these regions. Because these fragments have different homologous end sequences, they can be adapted into a single recombinant in yeast cells in a certain order. As shown in Figure 10, three large pieces of DNA were assembled in yeast into a total of 340 kbp recombinants containing part of the human IGL locus.
  • DNA fragment 1 was cloned from the BAC vector RP11-685C18, and after I-SceI digestion, it was about 38 kbp (GRCh37 / hg19Chr22: 22377208-22415482).
  • DNA fragment 2 (155 kb) was cloned from the BAC vector RP11-890G10, which is about 155 kb (GRCh37 / hg19Chr22: 22415353-22571119).
  • DNA fragment 3 was cloned from RP11-373H24 and was about 147 kb (GRCh37 / hg19Chr22: 22570833-22718740).
  • Competent yeast (ABI1380) was prepared using the above method. Competent yeast equivalent to 5 ml of the original yeast culture was incubated with 50 ng of linearized vector pTARYAC-HIS5 ( Figure 11) in a volume of 200 ⁇ l and 500 ng of exogenous gene fragments isolated from the above recombinant vector at 30 ° C. for 30 minutes, and then Heat shock in a 42 ° C water bath for 20 to 25 minutes. Centrifuge at 6000-8000r / min for 15 seconds, and remove the conversion mixture with a micropipette.
  • pTARYAC-HIS5 was obtained by transforming the vector pTARYAC-TRP1, in which the TRP1 gene was replaced in situ by the HIS5 gene.
  • the HIS5 gene sequence is shown in Sequence 2, SEQ ID NO: 2.
  • the selection of recombinants requires two rounds of PCR screening. The first round of screening was used to identify recombinant positive clones. Design primers (V-3F, V-5R) at both ends of the vector recombination site to amplify the self-circular vector. In addition, a third primer (D3-3F) was designed at the edge of the recombinant foreign fragment to amplify the recombinant. The primer sequence is shown in Figure 12 (shown below).
  • the electrophoresis bands 1-4 and 7-14 are negative controls, and the amplified products are self-circularized plasmids.
  • the electrophoretic bands 5, 6, and 15 are recombinant PCR products. Because the vector inserts a large fragment of foreign gene, the PCR product of the empty vector is negative, and the third primer on the foreign fragment can amplify another PCR band with another primer at the end of the vector. The second round of screening is used to identify whether the recombinants carry all foreign fragments. Design primers at the junctions of gene fragments and DNA fragments to identify positive clones.
  • the PCR primer sequence is shown in FIG. 12 of the specification, and FIG. 13B is the PCR identification result of the recombinant gene fragment. After assembly in yeast cells, the recombinant size reached 340kb.
  • the efficiency of recombination transformation is not only inversely proportional to the number of fragments, but also proportional to the molecular copy number of foreign genes (Gibson, Benders et al. 2008). Because the larger the DNA fragment, the lower the molar number of molecules. In order to increase the conversion efficiency of large molecular weight DNA, it is necessary to increase the template amount of the target fragment. However, the traditional DNA gel purification method is easy to cause the loss and fragmentation of large fragments of DNA, so the amount of template used for transformation will be less. For 100ug of 150kb DNA, after gel recovery, large fragments of DNA are easily broken and the recovery rate is extremely low. The recovery efficiency after each purification is less than 30ng. Figure 14 shows that the assembly efficiency of three large DNA fragments in yeast is compared. Increasing the amount of DNA template can increase the assembly efficiency of multiple large DNA fragments in yeast.
  • yeast bacterial shuttle plasmids we can amplify and purify a large number of foreign genes from bacteria for transformation.
  • yeast plasmids with different selectable markers (pTARYAC-HIS5) for recombination, see Figure 11.
  • the linear vector fragment pTARYAC-TRP1 will also be transferred into yeast cells, but it is easily degraded by the DNase of the yeast cell, or after self-cyclization, because of the activity of the URA3 gene expression, it is used by the drug -FOA kills.
  • Sequence 1 SEQ ID NO: 1
  • Sequence 7 SEQ ID NO: 7
  • Sequence 10 SEQ ID NO: 10
  • Sequence 11 SEQ ID NO: 11
  • Sequence 12 SEQ ID NO: 12
  • Sequence 13 SEQ ID NO: 13
  • Sequence 14 SEQ ID NO: 14
  • Sequence 15 SEQ ID NO: 15
  • Sequence 16 SEQ ID NO: 16
  • Sequence 17 SEQ ID NO: 17
  • Sequence 18 SEQ ID NO: 18
  • Sequence 19 SEQ ID NO: 19
  • Sequence 20 SEQ ID NO: 20
  • Sequence 21 SEQ ID NO: 21

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Abstract

一种高效，快速，简易地克隆目的基因片段的方法，尤其是克隆和编辑长度大于100kb，优选大于300kb的DNA大片段的方法。所述方法包括目的基因片段的获得、克隆、扩增、酶切和转化等过程。

Description

DNA大片段的克隆方法
本申请要求在2018年10月23日提交的，发明名称为“DNA大片段的克隆方法”，申请号为201811238618.6的优先权。
发明领域
本申请涉及基因工程技术领域。具体地，涉及克隆目的基因片段的方法，本申请的方法尤其适合克隆和编辑长度大于100kb，尤其300kb以上的DNA大片段。
发明背景
对真核和原核生物进行大规模的基因改造依赖大片段基因组DNA克隆技术。但是，目前尚缺少一种高效的克隆超大基因组DNA(一般大于100kb以上，尤其300kb以上)，尤其是真核基因组的方法。本专利介绍了一种高效的克隆和编辑超大基因片段，尤其是真核基因组片段的方法组合技术。
传统的基因文库法可以用于克隆一定长度的基因组DNA，例如细菌人工染色体文库最多只可以克隆300kb的外源DNA，酵母人工染色体(YAC)文库，可以克隆200kb到1000kb的基因(Dausset,Ougen et al.1992)。但是基因组文库的构建和阳性克隆的筛选需要大量的工作。另外，现有YAC文库不稳定，长期保存会发生嵌合体和基因丢失(Scott and Vos 2001)。
另一种方法是利用酵母转化相关重组克隆(TAR)技术选择性地从复杂和简单的基因组中克隆染色体大片段。但是，在现有技术中，TAR克隆技术较多地被用于克隆300kb以内的DNA(Kouprina and Larionov 2006)而很难用于来克隆超过300kb的基因。原因在于，300kb以上的基因只能从基因组DNA分离(BAC载体一般小于300kb)，随着被克隆基因变大，基因操作时DNA容易断裂，完整DNA浓度低，重组效率低。同时，这种方法依赖限制性内切酶分离DNA。当需要分离较大的DNA片段时，很难找到特异的内切酶完整的分割所需的DNA大片段。另外，在克隆较大的DNA片段时，TAR重组位点和DNA酶切位点较远也导致重组效率非常低，只有约2％。且这种重组效率随着DNA片段长度的增加而极度变低。
近年来发展的基因编辑技术(ZFNs(engineered zinc-finger nucleases),TALENs(transcription activator-like effector nucleases)and CRISPR-Cas9(clustered regularly interspaced short palindromic repeats that are recognized by Cas9nuclease)克服了上述缺点，有利于大片段基因的克隆。例如CRISPR/cas9系统，引导RNA(gRNA)可以特异性的识别20个核酸序列长度的DNA，而gRNA可以招募Cas9，在识别位点进行切割。跟限制性内切酶比，该技术可以具有极强的位点识别特异性。
现有CRISPR/cas9技术和DNA序列依赖的拼接技术(Ligationindependentcloning,LIC；Sequence and ligation-independentcloning，SLIC；Gibson assembling)联合，可将外源DNA重组到细菌人工染色体(BAC)(Jiang,Zhao et al.2015；Wang,Wang et al.2015)。然而，CRISPR和Gibson assembling技术联合的体外重组技术只能有效克隆100kb以下的DNA片段。
基因编辑技术还可以和细菌的Red/ET重组技术相结合用于克隆大片段的DNA。如，将被CRISPR切割的DNA片段也可和线性载体一起转化到表达λ噬菌体Red/ET重组系统的细菌中DNA片段可以在细菌体内通过序列依赖的酶促反应拼装成单一的重组子(Baker,Gupta et al.2016)。但是由于BAC载体装载能力和线性大片段DNA转化到细菌的限制，理论上也很难获得200kb以上的单个重组子。
酵母转化相关的重组(TAR)技术可以在酵母细胞内克隆20kb到250kb的外源DNA(Kouprina and Larionov 2006)。CRISPR/cas9技术和TAR克隆技术联合使用可以提高TAR克隆的效率但不能显著提高克隆大片段DNA的克隆长度(Lee,Larionov et al.2015)。并且由于YAC载体在酵母的拷贝数低，很难分离纯化以获得足量的DNA用于下一步克隆和基因编辑。在真核生物中，有些基因座或基因家族(如TCR、HLA、抗体基因、P450基因家族等)的长度要远大于300kb。目前尚无针对这些超大基因的克隆方法。
考虑到上述一步克隆方法不能得到超大基因片段的克隆，利用某些细胞特有的同源重组修复机制，有可能将多个基因片段在细胞内装配成超大片段的重组子。例如在表达lambda Red/ET重组酶的细菌可以实现细胞内多片段DNA的装配。但是，大多数真核基因富含重复序列，或GC富集区。由于细菌内重组酶活性，这些大片段基因序列容易在细菌内不稳定，易发生重组。另 外，细菌对于大片段DNA的转化能力低，也影响了这些大片段基因在细胞内的重组。有报道，枯草芽孢杆菌(Bacillus subtilis)可在细胞内装配大片段DNA(Itaya,Tsuge et al.2005；Yonemura,Nakada et al.2007)。但是枯草芽孢杆菌的培养不稳定，DNA转化能力低。也不适合多片段大基因的重组。现有技术中报道了，将DNA化学合成法，聚合酶链式反应(PCR)法，细胞外Gibson DNA装配法和酵母细胞同源重组依赖的多片段装配法联用，可将超过100万个碱基对的细菌基因克隆在YAC载体里(Gibson,Glass et al.2010)。但是，这种方法的缺陷在于DNA序列几乎是从头开始合成，操作步骤多，周期长，保真性差容，技术繁杂，成功率低。例如该方法需要很多步骤(基因合成，PCR，体外连接等方法联合使用)才能得到100kb的基因。而10个以上的100kbDNA片段才可以在酵母细胞内装配成超过1000kb的基因。
另外，真核生物的基因组DNA含有大量的简单重复序列。常规基因合成和PCR等方法很难对这些含有大量重复序列的真核生物DNA进行克隆。而这些DNA序列对基因表达的调节、基因组稳定性等具有重要的影响。因此从头合成大核酸装配法不仅技术繁琐步骤多而且还不利于含有简单重复序列的真核基因的克隆。综上，现有技术中尚缺少高效的，针对大片段基因组DNA，针对100kb以上，尤其300kb以上含有复杂序列真核基因克隆的方法。
此外，有文献报道，复制起始序列(autonomously replicating sequence，ARS)缺失的YAC载体可以有效地在酵母细胞中克隆含有类ARS序列的基因组DNA(Noskov,Koriabine et al.2001)。但是这种方法对目的基因有选择性。因此，需要建立新的YAC载体用于在酵母中克隆任何非选择性的大片段DNA。
发明概述
为了解决现有技术中存在的缺少针对大片段基因组DNA，特别是含有复杂序列的真核基因克隆的方法的问题，我们进行了深入的研究。
本申请首次提供了高效，快速，简易地克隆100kb以上，尤其300kb以上的DNA片段的方法。
具体来说，在本申请中，我们利用基因组编辑技术，例如Crispr/Cas9技术，TAR克隆技术和酵母DNA同源序列依赖的重组拼装技术相结合，快速实现大片段(100kb以上，尤其300kb以上)基因组DNA，特别是真核基因组DNA的编 辑和克隆。
另一方面，我们还设计了URA3基因依赖的阴性克隆负筛选策略，酶切后的大基因片段可以不必经过胶回收就可直接用于重组，含有空载体的酵母会被药物杀死，避免了载体的自身环化带来的假阳性。而归位内切酶的设计保证了大片段的切割的完整性。穿梭质粒保证了大基因片段在细菌的扩增。
虽然有文献报道，复制起始序列(autonomously replicating sequence，ARS)缺失的YAC载体可以用于含有类ARS序列的基因组DNA的克隆(Noskov，Koriabine et al.2001)。但是这种方法对目的基因有选择性。而利用URA3基因活性的负筛选法可以用于克隆任何真核基因和原核基因。
如上所述，相比现有技术中已知的DNA的克隆方法，例如基因合成，PCR等，本申请可以克隆含有简单重复序列的基因组DNA。相对于Crispr和Gibson assemble联用技术(不能克隆超过100kb的DNA片段,(Jiang,Zhao et al.2015))本申请利用Crispr/TAR克隆技术可以轻易制备一系列100kb到200kb的，甚至大于200kb的具有末端同源序列的DNA片段。相对于酵母TAR克隆技术,本申请可以实现多个100kb以上的基因在在酵母里装配成300kb以上的基因，因此，本申请的方法不仅能克隆超过300kb的DNA片段，而且还大大降低了克隆难度，增加了成功率。
此外，本申请的方法在克隆原核基因方面也比现有技术中的方法快速，高效。在酵母里拼装大片段的DNA的效率取决于转化到酵母的DNA片段数目和DNA片段的摩尔数量。本申请的方法利用酵母的TAR克隆技术可以容易地制备大量用于同源重组的大片段的DNA(100kb以上)，大大降低了用于拼装的DNA片段的数目。其次本申请方法利用了穿梭载体在微生物例如细菌大量扩增目的片段；利用归位内切酶完整切割目的大片段；利用URA3的负筛选系统，避免了载体的自身环化带来的假阳性；由于酶切产物可以直接用于转化，因此避免DNA胶回收后导致的DNA损失和DNA的断裂。以上设计保证了足够量的完整的大片段DNA用于酵母细胞的的装配，大大增加大片段DNA克隆的效率。综上，本申请首次提供了高效，快速，简易地克隆和编辑100kb以上，尤其300kb以上的DNA片段的方法。
具体地，本发明涉及以下方面：
在第一方面，本发明提供克隆目的基因片段的方法，其包括以下步骤：
1)从染色体或载体(优选BAC载体)获得目的基因片段；
2)在微生物细胞内将目的基因片段重组到另外的载体中；
3)使所述另外的载体在微生物中扩增；
4)用限制性内切酶消化所述另外的载体，以获得DNA片段；
5)将步骤4)获得的多个有同源末端的DNA片段和线性化的载体一起转化到有同源重组活性的微生物中，优选为酵母，从而在细胞内组装成获得包含多个目的基因片段的按照一定顺序排列的重组子。
在一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述步骤1)包括利用基因组编辑技术对所述染色体或载体进行切割以获得所述目的基因片段。
在一个优选的实施方案中，所述基因组编辑技术选自以下的一种或者多种：锌指核酸酶技术(zinc finger nucleases，ZFNs)，类转录激活因子效应物核酸酶技术(transcription activator-like effector nucleases，TALENs)和成簇的规律间隔的短回文重复序列(Clustered Regularly Interspaced Short Palindromic Repeat，CRISPR)技术包括CRISPR/Cas9和CRISPR-Cpf1，优选CRISPR/Cas9。
在一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述步骤2)包括利用同源重组拼接技术将所述目的基因片段克隆到所述另外的载体，优选穿梭载体中。
在一个优选的实施方案中，所述同源重组拼接技术选自以下的一种或者多种：序列依赖的体外装配技术(如Gibson assembling,SLIC，LIC等)，酵母的转化耦联重组(TAR)技术和细菌的Red/ET同源重组技术，优选酵母的转化耦联重组(TAR)技术。
在一个优选的实施方案中，在本申请提供的克隆目的基因片段的方法中，所述步骤4)中获得的DNA片段为真核基因片段，其为约100kb至300kb的DNA片段，并且所述DNA片段具有同源末端，优选60-800bp,更优选90-200bp的同源末端。
在一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述线性化的载体为拥有目的DNA片段同源末端序列的线性化载体，优选为线性化酵母穿梭载体。优选地，所述线性化的载体为YAC或TAR克隆载体。
在一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述 线性化的载体为YAC克隆载体，优选pTARYAC-TRP1克隆载体。
在一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述另外的载体选自以下的一种或者多种：BAC(bacterial artificial chromosome)、YAC(Yeast artificial chromosomes)和PAC(P1artificial chromosomes)，优选BAC载体。
在一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述酵母是酿酒酵母(saccharomyces cerevisiae)。
在一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述用于扩增的微生物是细菌，优选大肠杆菌，例如ElectroMAX TM DH5α-E TM Competent细胞(Invitrogen,Cata:11319019)。
在一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述目的基因片段包括原核和真核基因片段，优选为真核基因片段，优选其长度超过100kb，优选超过300kb,更优选超过400kb。
在另一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述限制性内切酶是归位内切酶，例如I-CeuI、I-SceI、PI-PspI、PI-SceI等。
在另一个实施方案中，在本申请提供的克隆目的基因片段的方法中，所述细胞是有DNA同源重组活性的细胞，优选酵母细胞，例如酿酒酵母(saccharomyces cerevisiae)。
在又一个优选的实施方案中，在本申请提供的克隆目的基因片段的方法中，所述方法还包括利用URA3抗性筛选系统筛选所述步骤5)获得的包含目的基因片段的重组子，所述筛选包括：
1)将所述目的基因片段整合到YAC载体中的ADH1启动子和URA3之间；
2)在含有5-FOA和相应营养缺陷型选择培养基，例如TRP1营养缺陷型选择培养基上筛选转化了包含所述目的基因片段的重组子的酵母细胞。
在再一个优选的实施方案中，在本申请提供的克隆目的基因片段的方法中，所述方法还包括在所述步骤4)和5)之间还包括以下步骤：重复步骤1)-4)以置备一系列的DNA片段，所述DNA片段具有同源末端，优选60-800bp,更优选90-200bp的同源末端。
在再一个优选的实施方案中，在本申请提供的克隆目的基因片段的方法中，所述方法还包括在步骤5)后再进行一次或多次步骤4)，获得多个具有同 源末端的DNA片段，优选100kb以上，将其再进行步骤5)而获得按照一定顺序排列的300kb以上的重组子。
在本申请的另一方面，本申请提供了一种利用URA3抗性筛选系统筛选重组了外源基因的YAC载体的方法，其包括以下步骤：
1)将外源基因整合到YAC载体中的ADH1启动子和URA3之间；
2)将步骤1)中获得的重组载体克隆到酵母细胞中；
3)在含有5-FOA和相应营养缺陷型选择培养基上筛选步骤2)中获得的含有重组载体的酵母细胞。
附图简述
图1.高效亚基因组克隆技术示意图，显示了基因编辑依赖的亚基因组快速DNA克隆技术。
图2.覆盖人IGL基因座(hg19chr22:22,385,572-23,265,082)的BAC载体。
图3.BAC载体上gRNA的设计与序列。
图4.gRNA结构示意图。在图4中，显示crRNA与tracrRNA配对。crRNA的可变靶特异性原型间隔区用“N”碱基表示。
图5.CRISPR/Cas9体外酶切结果。CRISPR/Cas9体外酶切外后通过DNA电泳鉴定基因组DNA和未携带靶DNA的载体。CRISPR/Cas9体外酶切及鉴定包括以下步骤：
步骤1.等摩尔tracrRNA和CrRNA混合形成gRNA；
步骤2.等摩尔gRNA和Cas9混合形成RNP复合体；
步骤3.RNP复合体细胞外酶切DNA模板；
步骤4.电泳鉴定。
在图5中，M表示DNA标志物，泳道1是Cas9 0ug，泳道2是Cas 9 0.125ug，泳道3是Cas 9 0.25ug，泳道4是Cas 9 0.5ug。
图6.用SnapGene TM产生的酵母细菌穿梭质粒pTARBAC-TRP1图谱。
图7A和图7B.TAR阳性克隆PCR筛选示意图。图7A中是引物设计示意图。图7B是PCR阳性克隆鉴定图。在图7B中，M表示100bp分子量，泳道1表示空质粒PCR产物，泳道2-4表示重组子阳性克隆。
图8.TAR和CRISPR/Cas9联用技术用于从BAC载体克隆目的基因片段。
图9.URA3负筛选法鉴定TAR重组阳性克隆。
图10.啤酒酵母依赖的多DNA片段装配原理。
图11.用SnapGene TM产生的酵母细菌穿梭质粒pTARBAC-HIS5的图谱。
图12.鉴定酵母内多片段DNA装配引物。
图13A和图13B.PCR法鉴定多片段酵母转化子。在图13A中M表示分子量对照物；泳道1-4,7-14表示空载体PCR产物；泳道5,6,15表示重组转化子PCR产物。在图13B中，泳道1表示载体和外源基因1连接处PCR产物；泳道2表示外源基因1的PCR产物；泳道3表示外源基因1和外源基因2连接处PCR产物；泳道4表示外源基因2的PCR产物；泳道5表示外源基因2和外源基因3连接处的PCR产物；泳道6表示外源基因3的PCR产物；泳道7表示外源基因和载体连接处的PCR产物；泳道8表示空载体对照；泳道M表示分子量对照物。
图14.不同量多个大片段DNA在酵母内装配效率的比较。
发明详述
除非另外定义，本文使用的所有技术和科学术语具有如本领域普通技术人员通常理解的相同意义。
所有专利、公开的专利申请、其他出版物、和来自GenBank和本文提到的其他数据库的序列，就相关的技术而言，通过引用以它们的整体并入。除非另外指出，所提供实施方式的实施将采用分子生物学等的常规技术，其在本领域技术人员的技术范围内。这些技术在文献中充分解释。见例如Molecular Cloning：A Laboratory Mannual，(J.Sambrook等，Cold Spring Harbor Laboratory，Cold Spring Harbor，N.Y.，1989)；Current Protocols in Molecular Biology(F.Ausubel等编辑，1987和最新的)；Essential Molecular Biology(Brown ed.，IRL Press 1991)；Gene Expression Technology(Goeddel编辑，Academic Press 1991)；Methods for Cloning and Analysis of Eukaryotic Genes(Bothwell等编辑，Bartlett Publ.1990)；Gene Transfer and Expression(Kriegler，Stockton Press 1990)；Recombinant DNA Methodology(R.Wu等编辑，Academic Press 1989)；PCR：A PracticalApproach(M.McPherson等，IRL Press at Oxford University Press 1991)；Cell Culture for Biochemists(R.Adams编辑，Elsevier Science Publishers 1990)；Gene Transfer Vectors for Mamalian Cells(Miller&M.Calos编辑，1987)； Mammalian Cell Biotechnology(M.Butler编辑，1991)；Animal Cell Culture(Pollard等编辑，Humana Press 1990)；Culture of Animal Cells，2nd Ed.(Freshney等编辑，Alan R.Liss 1987)。
如本文所用，“一个(a)”或“一个(an)”意思是“一个(one)”、“至少一个”或“一个或多个”。
如本文所用，“限制性内切酶位点”指被限制酶识别并切割的靶核酸序列。限制酶是本领域所熟知的。
如本文所用，术语“目的基因片段”是指需要克隆的靶DNA片段，可以是基因组片段或者人工合成的外源片段，也可以是完整基因。
术语“基因组”包括自然发生的基因组和合成基因组，并包括遗传改造基因组，比如之前在实验室和自然中不存在的基因组，其包括修饰的基因组和包含来自多于一个种类的核酸和/或部分基因组的杂交基因组。术语“基因组”包括细胞器基因组(例如，线粒体和叶绿体基因组)、自我复制生物的基因组(细胞基因组)，其包括原核和真核生物、真菌、酵母、细菌(例如，支原体)、古细菌、脊椎动物、哺乳动物和其他生物，和病毒基因组以及依靠宿主增殖的其他基因组。基因组还包括没有落在任何已知林奈(Linnean)分类中的生物和合成生物的那些。示例性基因组可以是微生物基因组，比如包括细菌和酵母在内的单细胞生物的基因组。
如上所述，现有技术中缺少针对100kb以上，尤其300kb以上的大片段基因组DNA，特别是含有复杂序列的真核DNA克隆的方法。
同时，真核基因含有许多简单重复序列，很难利用基因合成和PCR扩展的方法合成。对于长度超过100kb的基因，也很难找到特异的限制性内切酶将大片段基因分离出来。
为了解决此问题，本发明建立了一种高效克隆100kb以上，尤其300kb以上基因，特别是真核基因的方法。与已有方法不同，本申请的方法不仅高效，省略从头合成或PCR依赖的基因扩增的步骤，而且可以还可以克隆含有大量简单重复序列的真核生物基因。
本申请的方法利用基因编辑技术和同源序列依赖的DNA重组技术可以快速分离大的亚克隆基因片段。其次，这种方法也可以用于纯化含有简单重复序列的基因组DNA。最后，利用酵母的同源重组机制，多个100kb以上的外源 基因片段在酵母体内组装成长度多达2Mb的单个重组子。这种方法不仅可以用于克隆100kb以上，尤其300kb以上的真核基因组DNA，也同样可以高效地克隆相同长度的大片段的原核基因组或DNA病毒基因组,不仅可以用于克隆染色体上连续的基因家族或基因座，还可以将染色体不同位置的基因座编辑到一起。
因此，本申请是一种高效的大片段(100kb以上，尤其300kb以上)DNA的基因组克隆技术。简单来说，本申请的技术方案主要包括以下方面：1.利用基因组编辑技术对染色体或载体的特定DNA片段进行切割；2.利用微生物细胞特有的同源序列依赖的DNA重组原理将这些片段克隆到新的载体里；3利用穿梭载体特性在微生物里扩增，通过限制性内切酶，特别是归位内切酶，将100kb至300kbDNA片段从载体里纯化出来；4.重复以上方法置备一系列拥有60-800bp，优选90-200bp同源末端的DNA片段，将这些片段和线性酵母载体(YAC)一起转化到酿酒酵母里,利用酵母的同源重组特性得到长度超过300kb的重组子。
具体来说，本申请克隆目的基因片段的方法涉及以下步骤：
1)从染色体或载体(优选BAC载体)获得目的基因片段
在此步骤中，优选利用最近发展的基因编辑技术，高特异性地将大片段的基因从基因组或载体分离出来。
基因编辑技术包括锌指核酸酶技术(zinc finger nucleases，ZFNs)、类转录激活因子效应物核酸酶技术(transcription activator-like effector nucleases，TALENs)和CRISPR(clustered regularly interspaced short palindromic repeats，成簇的规律间隔的短回文重复序列)/Cas9技术。因为它们识别序列比较长，所以比限制性内切酶更容易特异性剪切较长的基因片段。而现有的基因编辑技术中，CRISPR/Cas9技术更为简单快速。除了经典的CRISPR/Cas9系统，近年发展的CRISPR-Cpf1也用来编辑富含AT区域的基因组。
并且CRISPR/Cas9体外酶切的方法可参见已有技术，亦可分为以下步骤：
1.等摩尔tracrRNA(100μM)和CrRNA(100μM)混合形成gRNA。95℃变性5分钟，随后室温复性30分钟。
2.在室温等摩尔gRNA和Cas9混合形成RNP复合体，保温10分钟。
3.RNP复合体细胞外酶切DNA模板,37℃反应2小时。
4.在65℃，蛋白酶K消化Cas9 10分钟。
5.电泳鉴定。
2)将目的基因片段克隆到另外的载体中
在此步骤中，优选利用同源序列依赖的重组拼接技术将目的基因片段克隆到另外的基因载体中，从而获得亚重组子。
序列依赖的重组拼接技术包括序列依赖的体外装配技术(如Gibson assembling,SLIC，LIC等)，酵母的转化耦联重组(TAR)技术和细菌的Red/ET同源重组技术，克隆到一些基因载体里，例如BAC(bacterial artificial chromosome),YAC(Yeast artificial chromosomes)和PAC(P1artificial chromosomes)。
3)亚重组子在微生物中的扩增
由于载体，尤其是基因载体，例如YAC载体有很强的同源重组特性有助于大片段基因在细胞内重组，但是YAC在酵母细胞内的拷贝数低，同时由于酵母细胞比细菌生长缓慢，YAC载体和线性酵母染色体很难在体外分离，大片段DNA容易断裂等原因，从酵母中纯化含有完整大片段DNA载体，例如YAC载体的技术难度高且产物的产量非常低。(Noskov,Chuang et al.2011)。
对此，可以利用基因载体，例如穿梭载体(包括pTARYAC)的特性，将携带有外源基因的酵母载体转化到非酵母微生物，优选细菌内，从而使获得的亚重组子可以在微生物，例如细菌中得到扩增。从细菌中可以大量制备300kb以内的载体DNA。已知DNA片段的大小可以影响其在微生物内的转化效率。例如在细菌转化240Kb质粒的效率比转化80Kb质粒DNA的效率低30倍。因此我们选用高效感受态菌株，例如ElectroMAX TM DH5α-E TM Competent细胞(Invitrogen,Cata:11319019)来转化DNA。
4)用限制性内切酶消化所述另外的载体，以获得DNA片段
其为约100kb至200kb的DNA片段，所述片段具有60-800bp,优选90-200bp同源末端。
通过载体里设计的限制性内切酶,消化所述另外的载体，以获得DNA片段。所述DNA片段为约100kb至300kb的DNA片段，并且所述DNA片段具有同源末端，优选60-800bp,更优选90-200bp的同源末端。
限制性内切酶优选的是归位内切酶。
由于克隆的基因组DNA序列比较大，不太容易找到合适的内切酶将大片段外源基因从载体完整的分离出来。
对此，我们在本申请的基因克隆方法中使用了归位内切酶。归位内切酶包括I-CeuI，I-SceI，PI-PspI，PI-SceI等，是双链DNA核酸酶，能识别较长的非回文序列(12-40bp)。归位内切酶的识别序列非常稀少。例如，18bp的识别序列在每7X10 10bp相当于20个哺乳动物基因组DNA的总长度中只出现一次。因此利用归位内切酶可以将任意的外源基因片段从载体分离出来。
也就是说，归位内切酶在基因组中的识别序列极少。根据此特点，使用归位内切酶可以将任意长度的目的片段从基因载体。例如pTARYAC-TRP1载体中完整的富集分离出来。
5)将以上获得多个DNA片段和酵母载体一起转化到酵母中，从而获得包含目的基因片段的重组子
将所述DNA片段，优选具有60-800bp,优选90-200bp同源末端的序列和酵母载体，优选线性酵母载体一起转化到酵母中，利用酵母的同源重组机制得到包含目的基因片段的重组子。
酵母作为宿主细胞包括酿酒酵母和其他酵母种类比如粟酒裂殖酵母(Saccharomyces pombe)，其可用于克隆超长度的基因组DNA。由于它们独特的遗传操作工具组，酵母宿主尤其适合操作供体基因组物质。酵母细胞的天然能力和几十年的研究已产生了丰富的用于在酵母中操作DNA的工具组。这些优点是本领域熟知的。例如，酵母，用它们丰富的遗传系统，可通过同源重组装配和重装配核苷酸序列，这是许多容易得到的生物不具备的能力。酵母细胞可用于克隆不能克隆入其他生物的更大的DNA，例如，整个细胞、细胞器和病毒基因组。因此，所描述方法的一种实施方式利用酵母遗传的巨大能力，通过使用酵母作为用于操作难处理的和其他生物的基因组和合成基因组的宿主细胞，推进合成生物学和合成基因组学。
另外，酵母，特别是酿酒酵母(Saccharomyces cerevisiae)作为克隆DNA片段的宿主细胞具有很大的优势。例如酵母有很强的重组酶活性，可以一次将多达25个重叠的片段的DNA片段连接到载体DNA上(Gibson,Benders et al.2008)，因此具有摄入多个DNA的能力。同时酵母的同源重组能力可以有效地将多片段DNA正确地装配为单个重组子。
另外，酵母人工染色体(Yeast artificial chromosome,YAC)含有着丝粒可以支持至少2Mb的真核不稳定DNA的复制(Kouprina,Leem et al.2003)。大的原核DNA片段可使用通用的遗传密码在酵母中克隆。有毒的基因表达通常不是在酵母中克隆供体核酸的障碍。例如，细菌和古生菌基因组的研究指示由于真核生物与这些细菌使用不同的蛋白表达机器，所以从克隆基因组表达的蛋白对酵母宿主具有很少的危害风险。在酵母中转录信号与在细菌中的转录和翻译不同。事实上，大部分原核基因很可能不在酵母中表达。在酵母中没有限制障碍。如果有障碍，那么它可能是复制障碍，而不是基因表达障碍。基因毒性被最小化，这是因为在真核生物比如酵母中的基因表达调节与在原核生物中的不同。而且，支原体使用密码子UGA用作色氨酸而不是作为翻译终止信号。因此，大部分支原体基因，如果表达的话，将在酵母中产生截短的蛋白质。这很大程度上避免了有毒基因产物的可能性。
因此，我们将这些含有同源序列末端的DNA片段和载体，例如基因载体，包括线形YAC载体一起导入酵母里。利用酵母特有的高效DNA同源重组机制，这些基因片段可以在酵母细胞内装配出环状的YAC载体。
一方面，载体包含在一个或多个期望细胞类型中促进载体复制所需的任何DNA元件(例如，复制原点)和用于不同细胞类型中的选择和/或抗性标记。
抗性标记是熟知的。技术人员能够为不同的宿主/供体组合确定合适的抗性标记。在一些情况下，期望使用非临床相关的标记。在其他情况下，抗性标记的选择取决于供体、宿主和/或受体细胞的性质。
酵母优选是酿酒酵母(saccharomyces cerevisiae)。
酵母的同源重组
酵母例如酿酒酵母存在高效的同源重组机制，两个DNA分子间只要有60bp以上的同源区段就可以准确、有效地进行同源重组(Noskov,Koriabine et al. 2001)。
本申请中利用基因编辑技术和酵母TAR克隆技术，可以产生一系列拥有同源末端的大基因片段。这些基因片段大小在约100kb到300kb之间，而彼此之间拥有同源末端，长度在60bp到800bp之间,优选长度在90bp到200bp之间。例如设计以下引物来扩增YAC穿梭载体pYACTAR-TRP1，从而用于克隆BAC载体(RP11-890G10)内部分人IGL基因座序列。引物序列的大写字母标示内切酶I-SceI的识别位点，I-SceI识别位点上游((60nt)，以小写字母标示)是BAC载体(RP11-890G10)内需要被克隆的目的基因的同源序列，I-SceI识别位点下游((30nt)，以小写字母标示)是pYACTAR-TRP1载体被扩增DNA的识别序列。通过PCR扩增pYACTAR-TRP1载体后，线性的载体的末端含有需要克隆DNA片段的同源序列。
用于扩增YAC穿梭载体pYACTAR-TRP1的正向引物(序列3：SEQ ID NO:3):
Figure PCTCN2019111498-appb-000001
用于扩增YAC穿梭载体pYACTAR-TRP1的反向引物(序列4：SEQ ID NO:4):
Figure PCTCN2019111498-appb-000002
真核基因组DNA存在许多重复简单序列，所以应避免在重复简单序列区域设计同源末端。由于这些片段同源末端序列不同，因此DNA片段可以按照一定顺序在酵母细胞内适配成单一的重组子。因此，利用酵母的同源重组机制，可以将具有同源末端的多个DNA片段进行同源重组，从而连接成单一的重组子，由此实现多个大片段DNA在酵母细胞中的拼接。
另外，利用酵母的TAR克隆技术，还可将线性的重组子可以通过线形YAC载体连接成环状的YAC重组子。这种环状的YAC载体含有着丝粒，可以将重组子在复制过程中分布于亲代细胞和子代细胞。另外环状结构可以避免线形外源DNA在酵母内被DNA酶水解。YAC载体表达啤酒酵母依赖的多片段装配原理见图10。
6)包括利用URA3抗性筛选系统筛选上述步骤5)获得的包含目的基因片段的重组子
在本申请的方法中，为了提高阳性重组子的筛选效率，我们还可以采用URA3抗性筛选系统筛选所述步骤5)获得的包含目的基因片段的重组子。
由于YAC载体，例如线性载体pTARYAC-TRP1在酵母细胞内容易自身环化，从而产生很多阴性克隆，这导致了很多假阳性结果。为了避免载体的自身环化带来的假阳性结果，我们设计了URA3抗性筛选系统，其中将5-FOA作为一种负筛选药物。
自身环化的载体在酵母细胞可表达URA3。URA3基因编码的酶可以使酵母细胞不能在含5-FOA的培养基上生长。有报道，当ADH1启动子的TATA盒与转录起始点之间的距离超过130bp时，ADH1启动子失去对下游基因的转录活性(Furter-Graves and Hall，1990)。而当外源基因被重组到ADH1启动子和URA3之间时，酵母细胞不表达URA3，因此对5-FOA不敏感可以在含有5-FOA的培养基上生长。这种筛选方法可以增加阳性重组子的筛选频率，统计结果如图9。
虽然有文献报道，复制起始序列(autonomously replicating sequence，ARS)缺失的YAC载体可以用于含有类ARS序列的基因组DNA(Noskov，Koriabine et al.2001)，但是这种方法对目的基因有选择性。而利用URA3基因活性的负筛选法可以用于克隆任何不含类ARS序列真核基因和原核基因。
同时本申请提供了以下实施例，以帮助理解本发明，在所附权利要求中给出了本发明的真正的范围。应当理解，在不背离本发明精神的情况下，可以对给出的方法进行修改。
实施例:利用BAC载体为模板，克隆人Lamda轻链基因座(IGL)。
1.包含人Lambda轻链基因座(IGL)的BAC载体的选择及其纯化。
人IGL基因座位于人22号染色体长臂1区1带2号亚带。因人单倍型不同，该基因座包括70到71个IGLV可变区基因，7到11个IGLJ连接区基因和7到11个IGLC保守区基因，每个连接区基因后是保守区基因。按照基因组版本 GRCh37/hg19，人IGL基因座在染色体中的起止位置在人22号染色体22,385,572到23,265,082之间，共879,511个碱基。根据UCSC基因组浏览器显示(https://genome.ucsc.edu/)，找出一些BAC载体覆盖以上区域。BAC载体的信息和起止位置显示在图2中。这些BAC购自BACPAC Resources Center(BPRC)，长度在160kb到200kb。每个相邻的BAC载体之间最少有几十kb的重叠区域。BAC载体DNA经BAC提取试剂盒，如Takara公司的NucleoBond Xtra BAC(Cata#：740436.25)，纯化，储存于4℃冰箱。
从BAC文库里分离基因组DNA的优点是，基因组目标片段(100kb到200kb)可以在细菌扩增，并且序列信息明确。另外，当使用CRISPR/Cas9系统进行酶切时，gRNA在BAC载体上的非特异性识别位点比全基因组要少，保障了切割特异性，减少了脱靶效应。
2.在BAC重叠区域设计gRNA的靶序列。
BAC基因组文库中相邻的BAC载体的重叠区域一般有十几或几十kb，为了产生一系列有一定同源区域(60bp到800bp)的DNA大片段，需要在基因组上相邻的BAC质粒的重叠区域内设计gRNA，利用Cas9酶切割将目标DNA从BAC质粒分离出来。在选择引导RNA识别位点时应避免简单反向重复区域，重叠区内gRNA靶序列的间距不超过1kb，以有利于在该区域内设计引物进行TAR克隆。利用UCSC基因组浏览器内嵌合的在线软件，如CRISPOR，设计gRNA的靶序列。其搜索的特征序列为[5’-G19nt-NGG]。在挑选gRNA时还需要利用软件，例如Blat search( http://genome.ucsc.edu/cgi-bin/hgBlat)去分析基因组潜在的脱靶序列，特别是避免在BAC载体内出现的脱靶序列。设计结果见图3。
3.利用CRISPR/Cas9对BAC载体进行体外酶切。
gRNA由CRISPR RNA(crRNA)与反式激活crRNA(trans-activating crRNA，tracrRNA)组成，crRNA的一部分序列能与tracrRNA配对，形成双链RNA结构；一部分序列与靶目标互补区域，以此识别靶序列。tracr RNA不仅可稳定crRNA，还可参与Cas9蛋白与DNA的结合与切割。
crRNA和tracrRNA的获得不仅可以利用包含T7启动子的模板DNA通过反 转录获得，也可以直接化学合成。例如从IDTdna化学合成Alt-R CRISPR crRNA和Alt-R CRISPR-Cas9tracrRNA(IDTdna，Cata#1072533)。其序列及结构如图4。
CRISPR/Cas9体外酶切方法如下：
1).将10μl 10μM CRISPR-Cas9crRNA和10μl 10μM tracrRNA混合。95℃预热5分钟，然后冷却到室温。
2).准备Cas9核糖核蛋白复合体(ribonucleoprotein，RNP)混合物：将crRNA和tracrRNA的混合物，与Cas9酶(够自NEB，#M0386)等摩尔混合，溶解到PBS缓冲液里，终体积为100μl。RNP混合物室温反应10分钟。
3).准备体外酶切反应：1μl Cas9RNP复合物，10xCas9Nuclease Reaction Buffer，100nM BAC DNA，7μl H 2O。37℃反应60分钟。最后，加1μL Proteinase K(20mg/mL)56℃10分钟消化Cas9，将BAC DNA从Cas9分离。电泳，鉴定切割效率。
图5为BAC质粒DNA(RP11-890G10)被gRNA/Cas9酶切结果。gRNA由CrRNA和tracrRNA组成。CrRNA的序列分别是：
IGL2A(rGrCrUrCrArCrUrGrGrGrGrCrCrArGrCrArGrCrUrGrUrUrUrUrArGrArGrCrUrArUrGrCrU)；IGL2B(rUrArUrCrCrUrArCrArGrGrArArUrArArCrArArCrGrUrUrUrUrAGrArGrCrUrArUrGrCrU)。tracrRNA购自IDTdna
(#1072533)。图5从左至右分别是1kb plusDNA ladder(NEB Cat#：N0552S和在不同Cas9酶当量作用下，BAC(RP11-890G10)被不同含量的CRISPR/Cas9复合物酶切结果。
4.TAR法克隆CRISPR/Cas9剪切后的目的基因
当BAC质粒上所需区域被CRISPR/Cas9体外酶切之后，分离出来的目的基因片段可以同线性的酵母细菌穿梭质粒(pTARYAC-TRP1)一起转化到酵母细胞中。在酵母细胞内，目的基因片段和线性的pTARYAC-TRP1通过各自末端的同源序列装配成环状的YAC载体。
穿梭质粒pTARYAC-TRP1(如图6)由pBACe3.6(购自BACPAC Resources Center，Cata#：pBACe3.6)改造而成。一部分DNA来自质粒pBACe3.6，包含细菌复制序列和选择性标记，如包括repE基因、sopA基因、sopB基因、oriT1复制子和选择性抗性基因氯霉素(Chloramphenicol resistant gene，CMR)；另一 部分DNA来自基因化学合成，包含酵母相关复制序列和选择抗性基因，如复制起始点序列ARS4，着丝点结构域CEN5，抗性基因TRP1，URA3等。合成的基因序列在序列1,SEQ ID NO:1中显示。酵母菌株AB1380可购自美国模式培养物集存库(
Figure PCTCN2019111498-appb-000003
20843)。其基因表型为MATa ade2-1lys2-1can1-100trp1 ura3his5。
线性化pTARYAC-TRP1载体的制备。
在pTARYAC-TRP1的ADH1启动子和URA3基因转录起始位点之间引入I-SceI位点。利用限制性内切酶I-SceI，将pTARYAC-TRP1线性化。设计引物用于扩增线性化的pTARYAC-TRP1载体，从而来克隆BAC载体(RP11-890G10)内的部分人IGL基因座序列。引物序列如下:
用于扩增YAC穿梭载体pYACTAR-TRP1的正向引物(序列3：SEQ ID NO:3):
Figure PCTCN2019111498-appb-000004
用于扩增YAC穿梭载体pYACTAR-TRP1的反向引物(序列4：SEQ ID NO:4):
Figure PCTCN2019111498-appb-000005
引物的3’端(以小写字母显示)，30bp左右，与线性化载体的末端相同。引物的5’端(小写字母显示)具有60个以上碱基，与BAC载体中需要克隆基因片段的末端序列相同。在引物5’和3’端之间(以大写字母显示)是内切酶I-SceI识别位点。
利用高效高保真扩增酶，例如TaKaRa LA
Figure PCTCN2019111498-appb-000006
DNA Polymerase(TaKaRa RR002A)，扩增经I-SceI线性化的pTARYAC—TRP1载体。扩增后，PCR产物末端将拥有同源序列用于重组目的片段。
TAR和CRISPR/Cas9联用技术用于从BAC载体克隆靶基因的方法如图8所示。将100ng经CRISPR/Cas9线性化的目的基因和100ng经PCR线性化的载体共转化到感受态酵母AB1380中。在液体YPAD培养基(Katara,Cata#：630306)培养24hr后，将酵母细胞涂抹在含有5-氟乳清酸(5-Fluoroorotic acid hydrate，5-FOA，Sigma F5013)和TRP选择性的固体合成培养基(Katara,Cata#：630309)上并培养。
酵母转化方法。
接种AB1380酵母(ATCC Cata#201447)在5mlYPAD培养液，30℃下振荡培养过夜。计数过夜培养物细胞密度，以最终5×10 6个/ml的细胞密度接种到50ml YPAD培养液。置30℃，200r/min振荡培养至2×10 7个细胞/ml。用50ml无菌离心管以3000g(2500r/min)离心5分钟，收获细胞。弃培养液，把细胞悬浮在25ml无菌水中，再同上离心.弃水。把细胞悬浮在1ml的100mmol/L醋酸锂中，转悬浮物到一个无菌1.5ml离心管。高速离心5秒钟沉淀细胞，用微量移液器吸出醋酸锂。悬浮细胞到最终500μl体积，其中大约含400μl的100mmol/L醋酸锂。振荡细胞悬浮液，取50μl酵母细胞到标记的离心管中，离心沉淀细胞，用微量取样器除去醋酸锂。基本“转化混合液”(Sigma Cata#YEAST1-1KT)由下列成分组成:240μl PEG(50％w/v)，36μl 1.0mol/L醋酸锂。在混合了25μl线性载体DNA(2.0mg/ml)，50μl经Cas9酶切线性化的DNA(0.1～10μg)后，剧烈振荡每个反应管直到细胞完全混匀，通常需要1分钟左右。30℃保温30分钟，然后置42℃水浴中热激20～25分钟。以6000～8000r/min离心15秒，用微量移液器除去转化混合液。吸01.0mlYPD液体培养基(Sigma Cata#Y1375)加到每个反应管中，用移液器轻轻悬浮沉淀细胞，30℃培养过夜。第二天，等份的200μl转化混合液均匀涂抹在含有0.5mg/mL 5-FOA的TRP缺失的合成培养基上(TAKARA，630309)。48小时后，挑取并扩增阳性克隆，以试剂盒(E.Z.N.A.
Figure PCTCN2019111498-appb-000007
Yeast DNA Kit，D3370-01)提取酵母DNA，利用PCR检测鉴定阳性克隆。在线性载体的末端相对方向设计两条引物用于扩增空载体，第三条引物设计在目的基因的末端同源重组序列的上游，用于扩增所插入的阳性克隆。引物设计见图7A。引物序列如下：用于扩增空载体的引物1(序列5：SEQ ID NO:5)：CATCAGCTCTGGAACAACGA；用于扩增空载体的引物2(序列6：SEQ ID NO:6)：GGCAACCAAACCCATACATC；用于扩增所插入的阳性克隆的引物3(序列7：SEQ ID NO:7)：AAAGGCTCAACAGGTTGGTG。PCR鉴定结果如图7B。条带1是空载体的PCR产物。条带2，3，4是阳性克隆的PCR产物。
由于线性载体pTARYAC-TRP1在酵母细胞内容易自身环化，从而产生很多阴性克隆，这导致了很多假阳性结果。为了避免载体的自身环化带来的假阳性结果，我们设计了URA3抗性筛选系统，其中将5-FOA作为一种负筛选药 物。自身环化的载体在酵母细胞可表达URA3。URA3基因编码的酶可以使酵母细胞不能在含5-FOA的培养基上生长。当ADH1启动子的TATA盒与转录起始点之间的距离超过130bp时，ADH1启动子失去对下游基因的转录活性(Furter-Graves and Hall 1990)。而当外源基因被重组到ADH1启动子和URA3之间时，酵母细胞不表达URA3，因此对5-FOA不敏感可以在含有5-FOA的培养基上生长。设计和筛选原理图见图8。URA3负筛选法鉴定TAR重组阳性克隆统计结果如图9。
由图9可以明显地看出，URA3基因活性的负筛选法可以显著地增加阳性重组子的筛选频率，筛选掉了77％的假阳性结果。利用URA3基因活性的负筛选法可以用于克隆任何真核基因和原核基因。
5.从细菌中纯化穿梭载体pTARYAC-TRP1中克隆的外源基因。
重组后的pTARYAC-TRP1载体可以在细菌中扩增。因为在PCR引物中间设计了归位内切酶，如I-SceI位点，而归位内切酶在基因组中的识别序列极低。因此归位内切酶可以将任意长度的目的片段从pTARYAC-TRP1载体中完整的富集分离出来。
从细菌中扩增目的基因的方法如下：
利用商业化试剂盒(E.Z.N.A.
Figure PCTCN2019111498-appb-000008
Yeast DNA Kit，Omega Bio-tek，D3370-01)提取酵母DNA。利用电转化的方法，将100ngDNA加入50μ感受态细菌ElectroMAX TM DH5α-E TM Competent细胞(Invitrogen,Cata#11319019)。电转化条件是1350V和5ms pulse。电转化后立即加入1ml SOC培养基(Invitrogen,Cata#15544034)，37℃培养一个小时。取100μl培养物接种到含氯霉素(15ug/ml)的LB培养皿，37℃培养过夜。第二天，挑选细菌克隆在含抗性的液体((15ug/ml氯霉素)LB中培养扩增。用QIAGEN Large-Construct Kit(Cat No./ID:12462)，可以从1升的培养基中纯化出大约100μg重组pTARYAC载体DNA。纯化的DNA经I-SceI内切酶37℃消化过夜，可以将外源基因片段和载体分离出来。
由于YAC载体在酵母内的拷贝数低，酵母比细菌生长缓慢，YAC载体和线性酵母染色体很难在体外分离，大片段DNA容易断裂等原因，从酵母中直接提取环状YAC载体的大小为不超过500kbp。因此可以利用穿梭载体 pTARYAC的特性，可将在酵母内克隆的基因转化到细菌内，并在细菌内扩增目的片段。
但是对于大片段的DNA，细菌转化效率会降低。例如在细菌转化240Kb质粒的效率比转化80Kb质粒DNA的效率低30倍。因为电转化方法比传统化方法转化效率至少高10倍，因此我们选用电转化方法转化大分子量DNA。ElectroMAX TM DH5α-E TM Competent细胞的转化效率是>1x 10 10克隆/μg。因为含有recA1mutation可以增加外源DNA的稳定性,所以认为其他含有recA1mutation的高效感受态细菌适合扩增含有大片段DNA的质粒。
由于克隆的基因组DNA序列比较大，这导致很难找到合适的内切酶将大片段外源基因从载体完整的分离出来。与之相比，归位内切酶，包括I-CeuI，I-SceI，PI-PspI，PI-SceI等，是双链DNA核酸酶，能识别较长的非回文序列(12-40bp)。并且，归位内切酶的识别序列非常稀有。例如，18bp的识别序列在每7X10 10bp相当于20个哺乳动物基因组DNA的总长度中只出现一次，因此载体中设计的归位内切酶可以将任意的外源基因片段从载体完整分离出来。
6.在酿酒酵母(Saccharomyces cerevisiae)中装配多片段的DNA。
酿酒酵母存在高效的同源重组机制，两个DNA分子间只要有60bp以上的同源区段就可以准确、有效地进行同源重组(Noskov,Koriabine et al.2001)。
利用基因编辑技术和酵母TAR克隆技术，可以产生一系列拥有同源片段末端的大基因片段。这些基因片段大小可在30kb到300kb之间，而彼此之间拥有同源末端，长度在90bp到200bp之间。真核基因组DNA存在许多重复简单序列，所以应避免在这些区域设计同源末端。由于这些片段同源末端序列不同，因此可以按照一定顺序在酵母细胞内适配成单一的重组子。如图10,三个大片段DNA在酵母中装配成包含部分人IGL基因座的总共340kbp的重组子。DNA片段1克隆自BAC载体RP11-685C18,经I-SceI酶切后为约38kbp(GRCh37/hg19Chr22:22377208-22415482)。DNA片段2(155kb)克隆自BAC载体RP11-890G10，为约155kb(GRCh37/hg19Chr22：22415353-22571119)。DNA片段3克隆自RP11-373H24，为约147kb(GRCh37/hg19Chr22：22570833-22718740)。
另外，利用酵母的同源重组机制，可以将多片段装配的重组子和线形YAC载体连接成环状的YAC重组子。这种环状的YAC载体含有着丝粒，可以将重组子在复制过程中分布于亲代细胞和子代细胞。另外环状结构可以避免线形外源DNA在酵母内被DNA酶水解。
利用上述方法制备感受态酵母(ABI1380)。将等同于5ml原始酵母培养物的的感受态酵母以200μl的体积与50ng线性化载体pTARYAC-HIS5(图11)，以及500ng从上述重组载体分离的外源基因片段30℃保温30分钟，然后置42℃水浴中热激20～25分钟。以6000～8000r/min离心15秒，用微量移液器除去转化混合液。吸01.0mlYPD液体培养基(Sigma Cata#Y1375)加到每个反应管中，用移液器轻轻悬浮沉淀细胞，30℃培养过夜。第二天，将等份的200μl转化混合液均匀涂抹在含有0.5mg/mL 5-FOA的HIS缺失的合成的固体培养基上(TAKARA，630313)。48小时后，挑取并扩增阳性克隆，在液体培养基SD/-His生长((TAKARA，630312)。以试剂盒(E.Z.N.A.
Figure PCTCN2019111498-appb-000009
Yeast DNA Kit，D3370-01)提取酵母DNA，利用PCR检测鉴定阳性克隆。
pTARYAC-HIS5是由载体pTARYAC-TRP1改造而获得的，其中TRP1基因被HIS5基因原位替代。HIS5基因序列在序列2，SEQ ID NO:2中显示。
重组子的筛选需要经过两轮PCR筛选。第一轮筛选用于鉴定重组阳性克隆。在载体重组位点的两端设计引物(V-3F，V-5R)用于扩增自身环状载体。另外在重组外源片段的边缘设计第三条引物(D3-3F)用于扩增重组子。引物序列见图12(显示如下)。
Figure PCTCN2019111498-appb-000010
Figure PCTCN2019111498-appb-000011
如图13A中所显示的，电泳条带1-4和7-14为阴性对照，扩增产物为自身环化的质粒。而电泳条带5，6，15为重组子PCR产物。因为载体插入了大片段的外源基因，因此空载体的PCR产物为阴性，而外源片段上的第三条引物可以和载体末端的另一条引物扩增出另一条PCR带。第二轮筛选用于鉴定重组子是否携带全部外源片段。分别在基因片段的衔接处和DNA片段的内部设计引物用于鉴定阳性克隆。说明书附图12中显示了PCR引物序列，图13B为多基因片段重组子的PCR鉴定结果。在酵母细胞内装配后，重组子大小达到340kb。
重组转化效率不仅与片段数目成反比，还与外源基因的分子拷贝数成正比(Gibson,Benders et al.2008)。因为DNA片段越大，分子摩尔数越低。为了增加大分子量DNA的转化效率，需要增加目的片段的模板量。然而，传统的DNA胶纯化法容易导致大片段DNA的丢失和断裂，因此用于转化的模板量就会少。对于100ug的150kbDNA，经过胶回收，大片段DNA容易断裂且回收率极低。每次纯化后回收效率小于30ng。图14中显示了三个大片段DNA在酵母内装配效率比较增加DNA模板量可以增加多个大片段DNA在酵母内的装配效率。
用酵母细菌穿梭质粒，我们可以从细菌中扩增和纯化大量的外源基因用于转化。另外经酶切后，为了避免胶回收后带来的损失，在酵母中进行多片段装配的时候，我们设计了有不同选择性标记的酵母质粒(pTARYAC-HIS5)用 于重组，见图11。而酶切后，线性载体片段(pTARYAC-TRP1)虽然也会被转入酵母细胞，但是易被酵母细胞的DNA酶降解，或在发生自身环化后，因为表达URA3基因的活性而被药物5-FOA杀死。
本申请涉及的序列如下：
1.序列1：SEQ ID NO:1
载体pYACTAR-TRP1的合成序列:
Figure PCTCN2019111498-appb-000012
Figure PCTCN2019111498-appb-000013
Figure PCTCN2019111498-appb-000014
2.序列2：SEQ ID NO:2
HIS5基因序列
Figure PCTCN2019111498-appb-000015
3.序列3：SEQ ID NO:3
用于扩增YAC穿梭载体pYACTAR-TRP1的正向引物
Figure PCTCN2019111498-appb-000016
4.序列4：SEQ ID NO:4
用于扩增YAC穿梭载体pYACTAR-TRP1的反向引物
Figure PCTCN2019111498-appb-000017
5.序列5：SEQ ID NO:5
用于扩增空载体的引物1
Figure PCTCN2019111498-appb-000018
6.序列6：SEQ ID NO:6
用于扩增空载体的引物2
Figure PCTCN2019111498-appb-000019
7.序列7：SEQ ID NO:7
用于扩增所插入的阳性克隆的引物3
Figure PCTCN2019111498-appb-000020
8.序列8：SEQ ID NO:8
用于重组子筛选的引物V-3F:
Figure PCTCN2019111498-appb-000021
9.序列9：SEQ ID NO:9
用于重组子筛选的引物D1-5R:
Figure PCTCN2019111498-appb-000022
10.序列10：SEQ ID NO:10
D1F:
Figure PCTCN2019111498-appb-000023
11.序列11：SEQ ID NO:11
Figure PCTCN2019111498-appb-000024
12.序列12：SEQ ID NO:12
D1-3F:
Figure PCTCN2019111498-appb-000025
13.序列13：SEQ ID NO:13
D2-5R:
Figure PCTCN2019111498-appb-000026
14.序列14：SEQ ID NO:14
D2F:
Figure PCTCN2019111498-appb-000027
15.序列15：SEQ ID NO:15
D2R:
Figure PCTCN2019111498-appb-000028
16.序列16：SEQ ID NO:16
D2-3F:
Figure PCTCN2019111498-appb-000029
17.序列17：SEQ ID NO:17
Figure PCTCN2019111498-appb-000030
18.序列18：SEQ ID NO:18
D3F:
Figure PCTCN2019111498-appb-000031
19.序列19：SEQ ID NO:19
Figure PCTCN2019111498-appb-000032
20.序列20：SEQ ID NO:20
D3-3F:
Figure PCTCN2019111498-appb-000033
21.序列21：SEQ ID NO:21
V-5R:
Figure PCTCN2019111498-appb-000034
22.序列22：SEQ ID NO:22
V-3F:
Figure PCTCN2019111498-appb-000035
23.序列23：SEQ ID NO:23
V-5R:
Figure PCTCN2019111498-appb-000036

Claims (19)

  1. 克隆目的基因片段的方法，其包括以下步骤：
    1)从染色体或载体(优选BAC载体)获得目的基因片段；
    2)在微生物细胞内将目的基因片段重组到另外的载体中；
    3)使所述另外的载体在微生物中扩增；
    4)用限制性内切酶消化所述另外的载体，以获得DNA片段；
    5)将步骤4)获得的多个有同源末端的DNA片段和线性化的载体一起转化到有同源重组活性的微生物中，优选为酵母，从而在细胞内组装成获得包含多个目的基因片段的按照一定顺序排列的重组子。
  2. 根据权利要求1的方法，其中所述步骤1)包括利用基因组编辑技术对所述染色体或载体进行切割以获得所述目的基因片段。
  3. 根据权利要求1或2的方法，其中所述步骤2)包括利用同源重组拼接技术将所述目的基因片段克隆到所述另外的载体，优选穿梭载体中。
  4. 根据前述权利要求中任一项的方法，其中所述步骤4)中获得的DNA片段为约100kb至300kb的DNA片段，并且所述DNA片段具有同源末端，优选60--800bp,更优选90-200bp的同源末端。
  5. 根据前述权利要求中任一项的方法，其中所述线性化载体为拥有目的DNA片段同源末端序列的线性化载体，优选为线性化酵母穿梭载体。
  6. 根据权利要求2-5中任一项的方法，其中所述基因组编辑技术选自以下的一种或者多种：锌指核酸酶技术(zinc finger nucleases，ZFNs)，类转录激活因子效应物核酸酶技术(transcription activator-like effector nucleases，TALENs)和成簇的规律间隔的短回文重复序列(Clustered Regularly Interspaced Short Palindromic Repeat，CRISPR)技术，包括CRISPR/Cas9和CRISPR-Cpf1，优选CRISPR/Cas9。
  7. 根据权利要求3-6中任一项的方法，其中所述同源重组拼接技术选自以下的一种或者多种：序列依赖的体外装配技术(如Gibson assembling,SLIC，LIC等)，酵母的转化耦联重组(TAR)技术和细菌的Red/ET同源重组技术，优选酵母的转化耦联重组(TAR)技术。
  8. 根据前述权利要求中任一项的方法，其中所述另外的载体选自以下的一种 或者多种：BAC(bacterial artificial chromosome)、YAC(Yeast artificial chromosomes)和PAC(P1artificial chromosomes)。
  9. 根据前述权利要求中任一项的方法，其中所述酵母是酿酒酵母(saccharomyces cerevisiae)。
  10. 根据前述权利要求中任一项的方法，其中所述用于扩增的微生物是细菌，优选大肠杆菌，例如ElectroMAX TMDH5α-E TMCompetent细胞。
  11. 根据权利要求5的方法，其中所述线性化载体是YAC或TAR克隆载体。
  12. 根据权利要求11的方法，其中所述线性化载体是YAC克隆载体，优选pTARYAC-TRP1克隆载体。
  13. 根据前述权利要求中任一项的方法，其中所述目的基因片段，包括原核和真核基因片段，优选为真核基因片段，其长度优选超过100kb，优选超过300kb,更优选超过400kb。
  14. 根据前述权利要求中任一项的方法，其中所述限制性内切酶是归位内切酶，例如I-CeuI、I-SceI、PI-PspI、PI-SceI。
  15. 根据前述权利要求中任一项的方法，其还包括利用URA3抗性筛选系统筛选所述步骤5)获得的包含目的基因片段的重组子，所述筛选包括：
    1)将所述目的基因片段整合到YAC载体中的ADH1启动子和URA3之间；
    2)在含有5-FOA和相应营养缺陷型选择培养基，例如TRP1营养缺陷型选择培养基上筛选转化了包含所述目的基因片段的重组子的酵母细胞。
  16. 根据前述权利要求中任一项的方法，其中在所述步骤4)和5)之间还包括以下步骤：重复步骤1)-4)以置备一系列的DNA片段，所述DNA片段具有同源末端，优选60-800bp，更优选90-200bp的同源末端。
  17. 根据前述权利要求中任一项的方法，其中所述细胞是有DNA同源重组活性的细胞，优选酵母细胞，例如酿酒酵母(saccharomyces cerevisiae)。
  18. 根据前述权利要求中任一项的方法，还包括在步骤5)后再进行一次或多次步骤4)，获得多个具有同源末端的DNA片段，优选100kb以上，将其再进行步骤5)而获得按照一定顺序排列的300kb以上的重组子。
  19. 利用URA3抗性筛选系统筛选重组了外源基因的YAC载体的方法，其包括以下步骤：
    1)将外源基因整合到YAC载体中的ADH1启动子和URA3之间；
    2)将步骤1)中获得的重组载体克隆到酵母细胞中；
    3)在含有5-FOA和相应营养缺陷型选择培养基上筛选步骤2)中获得的含有重组载体的酵母细胞。
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