WO2021068779A1 - 定点整合大片段外源dna的方法 - Google Patents

定点整合大片段外源dna的方法 Download PDF

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WO2021068779A1
WO2021068779A1 PCT/CN2020/118294 CN2020118294W WO2021068779A1 WO 2021068779 A1 WO2021068779 A1 WO 2021068779A1 CN 2020118294 W CN2020118294 W CN 2020118294W WO 2021068779 A1 WO2021068779 A1 WO 2021068779A1
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gene
expression cassette
resistance
loxp
sequence
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黄菁
杨波
卢娜
卢刚
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黄菁
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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    • C12N2800/00—Nucleic acids vectors
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    • C12N2800/00—Nucleic acids vectors
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    • C12N2999/007—Technological advancements, e.g. new system for producing known virus, cre-lox system for production of transgenic animals

Definitions

  • This application relates to the field of genetic engineering technology. Specifically, it relates to a method for site-specific integration of target gene fragments.
  • the method of the present application is suitable for efficient site-specific integration of target gene fragments, especially large fragments of foreign DNA above 300 kb, into the genome of cells.
  • Mammalian artificial chromosome is a cloning vector isolated from mammalian cells and constructed from the replication initiation region, telomeres and centromeres. The vector can be used to load foreign DNA fragments larger than 1000 kb.
  • MMCT microcell mediated chromosome transfer
  • MMCT microcell mediated chromosome transfer
  • Yeast artificial chromosomes is a vector that can clone the largest DNA fragments in artificial chromosomes, into which 100-2000 kb foreign DNA fragments can be inserted.
  • the yeast spheroplast cell fusion technology can be used to insert YAC vectors equipped with large fragments of foreign DNA into the chromosomes of recipient cells (Mendez, Green et al. 1997).
  • this method has a series of problems: the number of copies of foreign genes is uncontrollable, the location of genome integration is random, the YAC vector regulatory sequence causes the recipient cells to be contaminated with genes, and large fragments of genes are easily broken during cell fusion and integration.
  • both a large number of genotype identification of gene-edited ES cell clones and the identification of the phenotypes of different transgenic mouse lines are required.
  • Bacterial artificial chromosome is a bacterial chromosome cloning vector constructed on the basis of F-plasmid. It is commonly used to clone DNA fragments of 150kb-200kb in size. Because BAC vectors have the advantages of large capacity, genetic stability, and easy operation, they are often used for genetic modification. Using traditional homologous recombination technology or recombinase system, foreign genes ranging from 100 kb to 200 kb can be integrated into specific locations in the cell genome ((Valenzuela, Murphy et al. 2003; Wallace, Marques-Kranc et al. 2007). Subject to BAC The vector gene capacity is limited.
  • This patent solves the above-mentioned technical problems through a series of technological innovations, and provides for the first time a method for efficiently and stably integrating large fragments of foreign DNA, especially large fragments of foreign DNA over 300kb, into the genome.
  • the present application uses an improved recombinase system Cre/Loxp, that is, the Loxp71/66 mutant is used to stably and irreversibly integrate foreign DNA into the genome of the recipient cell.
  • Cre-Lox recombination is a site-specific recombinase technology used for deletion, insertion, transposition and inversion operations at specific sites in cell DNA.
  • the Loxp site is composed of a 34bp special site sequence.
  • the middle 8bp DNA base is an asymmetric sequence, which determines the direction of the Loxp sequence.
  • On both sides of the asymmetric sequence are two 13bp reverse symmetric sequences, which determine the binding efficiency with Cre.
  • Cre-Lox recombination method can catalyze the site-directed integration of foreign genes. But in the initial experiment, wild-type Loxp could not stably integrate macromolecular DNA into cell chromosomes. After analysis, this may be because the Loxp sequence-dependent recombination reaction catalyzed by Cre is reversible, that is, reverse recombination or deletion may occur between Loxp sites, so wild-type Loxp sites cannot effectively mediate the stable macromolecular DNA. Targeted integration into cell chromosomes.
  • Cre can still catalyze the recombination reaction between the mutant Loxp (such as Loxp71 and Loxp66). After Loxp71 and Loxp66 are recombined, wild-type Loxp and double mutant Loxp (Loxp71/66) are produced.
  • Loxp double mutation Loxp66/71 is the most preferred solution for mediating the stable and site-specific integration of macromolecular DNA into cell chromosomes.
  • Loxp with 1 mutation such as Loxp/Loxp66 or Loxp/Loxp71, or Loxp double mutation with other mutation positions
  • loxp66/71 has the best effect, the highest transformation efficiency, the most effective and stable Stable site-specific integration of macromolecular DNA into cell chromosomes, which is most suitable for site-specific integration and cloning of large fragments of DNA.
  • the present application also designs an expression cassette of a functionally complementary and truncated resistance gene neomycin (NeoR) after the Loxp sites of the DNA and the chromosome, respectively.
  • NeoR a functionally complementary and truncated resistance gene
  • this application uses the FLP/FRT system to cut out unnecessary vector sequences and G418 resistance expression cassettes.
  • the genetic modification elements and their recombination principles are shown in Figure 3 in the accompanying drawings of the specification.
  • this application selects YAC as a vector for loading foreign genes.
  • the traditional YAC vector has a linear structure, which is unstable in the cell and is prone to breakage and homologous recombination.
  • this application uses YAC vectors to load large fragments of DNA into circular YAC vectors, thereby improving the efficiency of site-specific insertion of complete genes into cell chromosomes.
  • the difficulty of targeted knocking of large fragments of DNA into recipient cells is that the transformation efficiency is low, the foreign DNA is easily broken, and the purification efficiency of large fragments of DNA from yeast cells is very low. Therefore, the yeast protoplast fusion technology is selected in this application. . Introduce large fragments of foreign genes from yeast into recipient cells. This method does not limit the size of the exogenous gene body, and can avoid the exogenous gene from breaking during the purification process (Brown, Chan et al. 2017).
  • This patent overcomes various problems in the process of site-specific integration of macromolecular DNA into chromosomal DNA through the above series of technical designs, such as poor transfection efficiency of macromolecular DNA, easy breakage of foreign genes, and poor site-specific integration efficiency. Therefore, the method of the present application can efficiently and stably integrate foreign DNA, especially large fragments of foreign DNA, more particularly large fragments of foreign DNA of more than 300 kb, into the genome at one time.
  • the present invention relates to the following aspects:
  • a method for integrating target gene fragments which includes the following steps:
  • the target gene fragment is cloned into the vector, and the resistance expression cassette 1, preferably truncated neomycin (G418) resistance expression cassette 1, is inserted into the obtained vector containing the target gene fragment, the expression cassette 1 Contains mutant Loxp 1 in the 5'to 3'direction, preferably Loxp66 sequence, part of HPRT intron and resistance gene a part, preferably neomycin resistance gene Neo gene 3'end part;
  • the vector containing the target gene fragment in the microorganism is introduced into the recipient cell, where the resistance expression cassette 2, preferably truncated neomycin (G418) resistance, has been introduced into the genome of the recipient cell.
  • Sexual expression cassette 2 contains part of HPRT introns, mutant Loxp 2, preferably Loxp71 sequence, resistance gene b part, preferably Neo gene 5'end part, containing female part in the 5'to 3'direction.
  • mutant Loxp 1 and the mutant Loxp 2 are respectively located in the 3'symmetric sequence and 5'symmetric sequence of the wild-type Loxp site, or the 5'symmetric sequence and 3'symmetric sequence of the wild-type Loxp site, Both the mutant Loxp 1 and the mutant Loxp 2 can bind to Cre, and after the step 4), a Loxp sequence containing mutations at both ends, such as Loxp66/71, is formed;
  • mutant Loxp 1 and mutant Loxp 2 do not affect the binding efficiency to Cre, and form two loxp sequences after recombination, one of which is a wild-type Loxp sequence, The other is a Loxp sequence with mutations at both ends, such as Loxp66/71, which reduces the binding efficiency to Cre compared to the wild-type Loxp sequence;
  • Loxp66 may be recombined with loxp71 to form a double-terminal mutant Loxp structure including loxJTZ17, loxKR1, loxKR2, loxKR3, loxKR4, etc.
  • Loxp structures mediate recombination in different cells, and the efficiency of integration into chromosomes to form stable recombinants is slightly different.
  • the efficiency of integration into the chromosome to form stable recombinants is affected by many factors, such as the integrated cell line.
  • the Loxp66/71 structure of the present application has the highest efficiency in forming stable recombinants in the chromosome of the cell, so the cloning efficiency of large DNA fragments is the highest;
  • CreERT2 Induce the recombinase activity of CreERT2, preferably by adding Tamoxifen to induce the recombinase activity of CreERT2, the Cre recombinase mediates the targeted integration of the target gene fragment into the genome of the recipient cell, and the expression cassette 1 And expression cassette 2 to form a complete resistance gene, preferably neomycin resistance expression cassette;
  • neomycin resistance protein can be expressed only after some recombination in a certain order.
  • the 5'end of the Neo gene and the 3'end of the Neo are truncated at position 92, and more preferably, the 92nd amino acid of neomycin is derived from the 5'end of the Neo gene.
  • the last nucleotide and the start nucleotide of the 3'end of the Neo gene are co-encoded.
  • the 92nd amino acid of neomycin is composed of the last 2 nucleosides of the 5'end of the Neo gene. The acid and the first nucleotide at the beginning of the 3'end of the Neo gene are co-encoded.
  • the 5'end of the Neo gene expresses the amino acids from 1 to 91 of neomycin
  • the 3'end of the Neo gene expresses the amino acids from 93 to 267 of neomycin
  • the 5'end of The 2 nucleotides at the end of and the first nucleotide at the beginning of the 3'end of the Neo gene together encode the 92nd amino acid of neomycin
  • step 6 by adding a reagent specific to the resistance gene, preferably the antibiotic G418, to screen the recipient cells in the genome that have integrated the target gene fragment at a specific point.
  • a reagent specific to the resistance gene preferably the antibiotic G418, to screen the recipient cells in the genome that have integrated the target gene fragment at a specific point.
  • step 6 it also includes the step 7): the plasmid containing FLP is transferred into the recipient cell to remove the expression cassette.
  • the target gene fragment in step 1) includes prokaryotic and eukaryotic gene fragments, preferably eukaryotic gene fragments, the length of which is preferably more than 100 kb, preferably more than 300 kb, more preferably more than 400kb.
  • the vector in step 2) is a linearized vector
  • the linearized vector is preferably a linearized vector having a homologous end sequence of the target DNA fragment to be cloned, and more Preferably, it is a linearized yeast shuttle vector.
  • the linearization vector is a YAC cloning vector, preferably a pTARYAC cloning vector.
  • step 2 9. The method according to one of items 1-5, wherein the vector containing the target gene fragment in step 2) is BAC (bacterial artificial chromosome) or PAC (P1 artificial chromosomes).
  • BAC bacterial artificial chromosome
  • PAC P1 artificial chromosomes
  • the circular vector is spliced by homologous recombination technology (preferably yeast transformation coupled recombination (TAR) technology), or homologous recombination splicing technology (preferably yeast transformation coupled recombination ( TAR) technology) and genome editing technology.
  • homologous recombination technology preferably yeast transformation coupled recombination (TAR) technology
  • homologous recombination splicing technology preferably yeast transformation coupled recombination (TAR) technology
  • homologous recombination splicing technology is selected from one or more of the following: sequence-dependent in vitro assembly technology (such as Gibson assembling, SLIC, LIC, etc.), yeast transformation coupled recombination ( 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 assembling, SLIC, LIC, etc.
  • yeast transformation coupled recombination ( TAR) technology and bacterial Red/ET homologous recombination technology, preferably yeast transformation coupled recombination (TAR) technology.
  • the target gene fragment in step 1) is obtained from a chromosome or another vector (preferably a BAC vector).
  • step 13 The method according to one of the preceding items, wherein the target gene fragment in step 1) is obtained by cutting the chromosome or vector using genome editing technology.
  • step 4 the expression cassette and its downstream large fragments of DNA are introduced into the genome of the recipient cell in a targeted and stable manner by genome editing technology.
  • the genome editing technology is selected from one or more of the following: zinc finger nuclease technology (zinc finger nucleases, ZFNs), transcription activator-like effector nuclease technology Transcription activator-like effector nucleases (TALENs) and clustered regularly spaced short palindromic repeat (Clustered Regularly Interspaced Short Palindromic Repeat, CRISPR) technologies include CRISPR/Cas9 and CRISPR-Cpf1, preferably CRISPR/Cas9.
  • yeast is Saccharomyces cerevisiae.
  • step 4 the yeast protoplast fusion technology is used to introduce the vector containing the target gene fragment in the microorganism into the recipient cell.
  • the resistance expression cassette 1 in step 2) is preferably the mutant Loxp 1 and/or the truncated neomycin (G418) resistance expression cassette 1
  • the resistance expression cassette 2 in the step 4), preferably the mutant Loxp 2 in the truncated neomycin (G418) resistance expression cassette 2 is used to introduce the expression cassette into the genome of the recipient cell. in.
  • the resistance expression cassette 1 in step 2) is preferably the HPRT intron and resistance gene contained in the truncated neomycin (G418) resistance expression cassette 1 Part a, preferably the 3'end of the Neo gene and the resistance expression cassette 2 in step 4), preferably the HPRT intron and resistance gene b contained in the truncated neomycin (G418) resistance expression cassette 2 Part, preferably the 5'end part of the Neo gene, and the resistance expression cassette expressing the complete resistance gene formed in step 5), preferably the neomycin (G418) resistance expression cassette of Neomycin Neo, for screening the target gene Cellular recombinant after site-directed integration of fragments.
  • the resistance expression cassette 1 in step 2 is preferably the HPRT intron and resistance gene contained in the truncated neomycin (G418) resistance expression cassette 1 Part a, preferably the 3'end of the Neo gene and the resistance expression cassette 2 in step 4), preferably the HPRT intron and resistance gene b contained in the truncated
  • the recipient cell in step 4 is a eukaryotic cell, preferably an animal embryonic stem cell.
  • this application can also use other resistance screening mechanisms known in the art.
  • These commonly used resistance screening mechanisms or resistance genes can be, for example, Puromycin, Hygromycin, HPRT, and the like.
  • Figure 1 Shows the overall strategy for site-specific integration of large fragments of foreign genes into the cell genome.
  • Loxp71/66 mutant promotes the targeted integration of macromolecular circular DNA.
  • Figure 3 Schematic diagram of the site-specific integration of exogenous circular DNA into cell chromosomes.
  • M means 100bp DNA ladder
  • Lane 1 is PCR product 1 at the junction between the end of the vector TAR-Loxp66 and IGL1,
  • Lane 2 is the internal PCR product 2 of IGL1,
  • Lane 3 is the PCR product 3 at the junction of IGL1 and IGL2,
  • Lane 4 is the internal PCR product 4 of IGL2,
  • Lane 5 is the PCR product 5 at the junction of IGL2 and IGL3,
  • Lane 6 is the internal PCR product 6 of IGL3, and
  • Lane 7 is the PCR product 7 at the junction of IGL3 and the end of the vector TAR-Loxp66.
  • Figure 6 The primer sequence used to identify TAR-IGL-Loxp66.
  • FIG. 1 Gene knock-in mediated by CRISPR/Cas9 technology.
  • FIG. 10 The 5'end of Neo gene in truncated neomycin (G418) resistance expression cassette 2 and the neomycin resistance gene Neo contained in truncated neomycin (G418) resistance expression cassette 1 Schematic diagram of the nucleotide sequence and amino acid sequence of the 3'end portion. In the figure, the 5'end of the Neo gene and the 3'end of the Neo are truncated at position 92, and the 5'end of the Neo gene expresses the amino acids from positions 1 to 91 of neomycin, and 3 of the Neo gene.
  • The'end part expresses the amino acids from position 93 to 267 of neomycin, and the 2 nucleotides at the end of the 5'end part and the first nucleotide at the beginning of the 3'end part of the Neo gene jointly encode the new Amino acid R at position 92 of mycin.
  • the unshaded part at the upper end shows that the 5'end of the Neo gene expresses the amino acid sequence of neomycin from position 1 to 91 and the corresponding nucleotide sequence
  • the gray shaded part at the lower end shows Neo.
  • the 3'end of the gene expresses the amino acid sequence of neomycin from position 93 to position 267 and the corresponding nucleotide sequence.
  • the black shaded part shows the 92th amino acid R, which is coded by the 2 nucleotides at the end of the 5'end, AG, and the first nucleotide G at the beginning of the 3'end of the Neo gene. of.
  • restriction endonuclease 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, which can be a genomic fragment or an artificially synthesized exogenous fragment, or a complete gene.
  • the term “genome” includes naturally occurring genomes and synthetic genomes, and includes genetically modified genomes, such as genomes that did not exist in the laboratory and in nature before, including modified genomes and containing nucleic acids and/or parts from more than one species The hybrid genome of the genome.
  • the term “genome” includes organelle genomes (e.g., mitochondrial and chloroplast genomes), genomes of self-replicating organisms (cell genomes), which include prokaryotic and eukaryotic organisms, fungi, yeast, bacteria (e.g., mycoplasma), archaea, vertebrates , Mammals and other organisms, and viral genomes and other genomes that rely on the host to multiply.
  • the genome also includes those of organisms and synthetic organisms that do not fall into any known Linnean classification.
  • Exemplary genomes can be microbial genomes, such as the genomes of single-celled organisms including bacteria and yeast.
  • the prior art lacks a method for site-specific integration of large fragments of genomic DNA above 300 kb in the genome.
  • the present invention establishes a method for efficiently and stably integrating genes of more than 300kb, especially eukaryotic genes, into the genome.
  • the method of the present application can not only stably integrate large foreign DNA fragments, such as 300 kb or more, into the genome, but also improve the efficiency of site-specific integration.
  • site-specific integration As mentioned above, large-scale DNA modification of the cell genome, especially the site-specific integration of large fragments of foreign DNA into the genome, has always been a difficult problem in current biotechnology.
  • the main technical difficulties of site-specific integration include the following: 1. How to clone large fragments of foreign DNA into gene vectors; 2. How to completely introduce foreign DNA-containing vectors into eukaryotic cells; 3. How to efficiently transfer large fragments of foreign DNA into eukaryotic cells The fragments of foreign DNA are integrated into the genome of the recipient cell at a specific site; 4. How to efficiently screen and detect the clones of the site-specific integration recombinant cells.
  • this application has all been designed to efficiently and stably integrate foreign DNA into the genome in a targeted manner. details as follows:
  • vectors especially gene vectors, such as YAC vectors
  • YAC vectors have strong homologous recombination characteristics, they can facilitate the recombination of large fragments of foreign DNA in cells.
  • this application selects YAC as a vector for loading foreign genes.
  • the traditional YAC vector has a linear structure, which is unstable in the cell and is prone to breakage and random recombination.
  • this application uses YAC vectors to load large fragments of DNA into circular YAC vectors, thereby improving the efficiency of site-specific insertion of complete genes into cell chromosomes.
  • homologous recombination splicing technology or a combination of homologous recombination splicing technology and genome editing technology is used to obtain a circular YAC vector containing the target gene fragment.
  • homologous recombination splicing technology such as yeast transformation-associated recombination (TAR) technology, is used to circularize the linear YAC vector into a circular closed YAC vector.
  • a combination of homologous recombination splicing technology and genome editing technology such as Crispr/Cas9 method and TAR technology, edit gene fragments in multiple BAC vectors into multiple linear DNA fragments with homologous ends , And use the characteristics of homologous recombination of Saccharomyces cerevisiae to splice into a circular YAC vector in its cells.
  • homologous recombination splicing technology such as Crispr/Cas9 method and TAR technology
  • Sequence-dependent recombination splicing technology includes sequence-dependent in vitro assembly technology (such as Gibson assembling, SLIC, LIC, etc.), yeast transformation coupled recombination (TAR) technology and bacterial Red/ET homologous recombination technology, cloned into some gene vectors
  • sequence-dependent in vitro assembly technology such as Gibson assembling, SLIC, LIC, etc.
  • yeast transformation coupled recombination (TAR) technology and bacterial Red/ET homologous recombination technology, cloned into some gene vectors
  • BAC bacterial artificial chromosome
  • YAC Yeast artificial chromosomes
  • PAC P1 artificial chromosomes
  • Yeast as a host cell includes Saccharomyces cerevisiae and other yeast species such as Saccharomyces pombe, which can be used to clone ultra-long genomic DNA. Because of their unique genetic manipulation tool set, yeast hosts are particularly suitable for manipulating donor genome 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, with their rich genetic system, can assemble and reassemble nucleotide sequences through homologous recombination, which is a capability that many readily available organisms do not possess. Yeast such as Saccharomyces cerevisiae has an efficient homologous recombination mechanism.
  • 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 ability of yeast inheritance to advance synthetic biology and synthetic genomics by using yeast as a host cell for manipulating the genomes and synthetic genomes of intractable and other organisms.
  • yeast especially Saccharomyces cerevisiae
  • yeast has great advantages as a host cell for cloning DNA fragments.
  • yeast has strong recombinase activity and can ligate up to 25 overlapping DNA fragments to carrier DNA at a time (Gibson, Benders et al. 2008), so it has the ability to take in multiple DNAs.
  • the homologous recombination ability of yeast can effectively assemble multiple fragments of DNA into a single recombinant.
  • Yeast artificial chromosome contains centromeres that can support at least 2Mb of eukaryotic unstable DNA replication (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 acid 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 the yeast host. The transcription signal in yeast is different from the transcription and translation in bacteria. In fact, most prokaryotic genes are probably not expressed in yeast. There are no restriction barriers in yeast.
  • a barrier may be a replication barrier, not 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 Mycoplasma genes, if expressed, will produce truncated proteins in yeast. This largely avoids the possibility of toxic gene products.
  • this application is based on the yeast-specific high-efficiency DNA homologous recombination mechanism, using YAC vectors in yeast cells to load large fragments of DNA into circular YAC vectors, thereby improving the efficiency of site-specific insertion of complete genes into cell chromosomes.
  • the difficulty of targeted knocking of large fragments of DNA into recipient cells is that the transformation efficiency is low, the foreign DNA is easy to break, and the efficiency of purifying large fragments of DNA from yeast cells in vitro is very low, and common DNA transfection methods also have limitations on the size of DNA. .
  • this application adopts a yeast protoplast fusion method to introduce large fragments of foreign genes in yeast into recipient cells.
  • yeast spheroplasts Under the mediation of PEG, yeast spheroplasts can fuse with cells, and large fragments of DNA are introduced into recipient cells.
  • This method does not limit the size of the exogenous gene body, and can avoid the exogenous gene from breaking during the purification process (Brown, Chan et al. 2017).
  • This application uses an improved recombinase system Cre/Loxp, that is, the Loxp71/66 mutant is used to stably and irreversibly integrate foreign DNA into the genome of the recipient cell.
  • the phiC31-att recombination system can mediate irreversible recombination reactions between DNA fragments, the activity of phiC31 is lower than that of Cre recombinase, so it is not the preferred solution for mediating the recombination of macromolecular DNA.
  • Cre/Loxp system has the highest catalytic efficiency and is the most commonly used. Therefore, this application considers using the Cre/Loxp system to mediate the recombination of large fragments of DNA.
  • Cre-Lox recombination is a site-specific recombinase technology used for deletion, insertion, transposition and inversion operations at specific sites in cell DNA.
  • the Loxp site is composed of a special sequence of 34bp.
  • the middle 8bp DNA base is an asymmetric sequence, which determines the direction of the Loxp sequence.
  • On both sides of the asymmetric sequence are two 13bp reverse symmetric sequences, which determine the binding efficiency with Cre.
  • Cre-Lox recombination can catalyze the site-directed integration of foreign genes. But in the initial experiment, it was found that the wild-type Loxp sequence failed to stably integrate the macromolecular DNA into the cell chromosomes. This may be because the Loxp sequence-dependent recombination reaction catalyzed by Cre is reversible, that is, reverse recombination or deletion may occur between Loxp sites, so the recombination between wild-type Loxp sites cannot effectively mediate the stability of macromolecular DNA Site-specific integration into cell chromosomes.
  • the present application uses tamoxifen to induce recipient cells, such as ES cells, to express Cre recombinase.
  • recipient cells such as ES cells
  • Cre recombinase When one end of the symmetric sequence in the Loxp sequence is mutated, that is, the mutant Loxp 1, preferably Loxp71 (the symmetric sequence at the 5'end has a mutation) and the mutant Loxp 2, preferably Loxp66 (the symmetric sequence at the 3'end has a mutation).
  • the mutations in the mutant Loxp 1 and mutant Loxp 2 are respectively located in the 3'symmetric sequence and 5'symmetric sequence of the wild-type Loxp site, or the 5'symmetric sequence and 3'symmetric sequence of the wild-type Loxp site. Sequence, the mutant Loxp 1 and mutant Loxp 2 can both bind to Cre, and after the step 4), a Loxp sequence containing mutations at both ends is formed, such as Loxp66/71;
  • mutant Loxp 1 and mutant Loxp 2 do not affect the binding efficiency to Cre, and form two loxp sequences after recombination, one of which is a wild-type Loxp sequence, The other is a Loxp sequence with mutations at both ends, such as Loxp66/71, which reduces the binding efficiency to Cre compared to the wild-type Loxp sequence;
  • Loxp66/71 structure of the present application has the highest efficiency in forming stable recombinants in the chromosomes of cells, and therefore the cloning efficiency of large DNA fragments is the highest.
  • the single-ended mutant loxp sequence, the mutant Loxp 1 and the mutant Loxp 2, preferably Loxp71 and Loxp66 do not affect the binding efficiency to Cre, and two loxp sequences are formed after recombination, one of which is The wild-type Loxp sequence, the other is a Loxp sequence with mutations at both ends, preferably Loxp66/71, which reduces the binding to Cre compared to the wild-type Loxp sequence.
  • the circular DNA can be inserted into the chromosome at a specific point and produce wild-type Loxp sites and Loxp 1/2 mutants, such as Loxp71/66 mutants, on both sides of the inserted fragment.
  • Loxp 1/2 type mutants preferably Loxp71/66 mutants
  • both ends of the symmetric sequence are mutated, and their ability to bind to Cre is greatly reduced, so Cre cannot effectively catalyze Loxp 1/2 type mutants, preferably Loxp71
  • the /66 mutant undergoes reverse recombination, resulting in large fragments of circular DNA that can be stably and irreversibly integrated into the cell chromosomes.
  • Loxp 1/2 mutants preferably Loxp71/66 mutants
  • Loxp1/2 mutants preferably Loxp71/66 mutants
  • reverse recombination reaction occurs, resulting in the stability of foreign DNA
  • the ground is irreversibly integrated into the cell chromosomes, thereby enhancing the stability of the inserted gene.
  • the wild-type Loxp site cannot effectively mediate large circular DNA into the chromosome by Cre-mediated stable site-specific integration.
  • the working principle of the Loxp71/66 mutant promoting the directed integration of macromolecular circular DNA is shown in Figure 2 in the accompanying drawings of the specification.
  • mutant Loxp such as Loxp71/66 mutant instead of wild-type Loxp, helps to efficiently and stably integrate large fragments of DNA into the cell genome.
  • Loxp double mutation Loxp66/71 is the most preferred solution for mediating the stable and site-specific integration of macromolecular DNA into cell chromosomes.
  • Loxp with 1 mutation such as Loxp/Loxp66 or Loxp/Loxp71, or Loxp double mutation with other mutation positions
  • loxp66/71 has the best effect, the highest transformation efficiency, the most effective and stable Stable site-specific integration of macromolecular DNA into cell chromosomes, which is most suitable for site-specific integration and cloning of large fragments of DNA.
  • the vector generally contains any DNA elements necessary to promote replication of the vector in one or more desired cell types (e.g., origin of replication) and selection and/or resistance markers used in different cell types.
  • Resistance markers are well known. The skilled person is able to determine the appropriate 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 application in order to efficiently screen the recombinants that are integrated into the chromosome, the application also designs a functionally complementary and truncated resistance gene neomycin (NeoR) expression cassette after the Loxp site of the DNA and chromosome. .
  • NeoR functionally complementary and truncated resistance gene neomycin
  • G418 functionally complementary and truncated resistance gene
  • this expression cassette can be used to identify site-directed insertions of foreign genes.
  • Any single truncated neomycin (G418) resistance expression cassette cannot make cells resistant to neomycin.
  • the 5'end portion of the Neo gene and the 3'end portion of the Neo gene are truncated at position 92. More preferably, the 92nd amino acid of neomycin is co-encoded by the last nucleotide of the 5'end portion of the Neo gene and the start nucleotide of the 3'end portion of the Neo gene. In a specific embodiment, the 92nd amino acid of neomycin is composed of the 2 nucleotides at the end of the 5'end of the Neo gene and the first nucleoside at the beginning of the 3'end of the Neo gene. Acid co-coded.
  • the 5'end of the Neo gene expresses the amino acids from 1 to 91 of neomycin
  • the 3'end of the Neo gene expresses the amino acids from 93 to 267 of neomycin
  • the 5'end of The two nucleotides at the end of and the first nucleotide at the beginning of the 3'end of the Neo gene jointly encode the 92nd amino acid of neomycin.
  • CreERT2-IRES-PuroR structure was also designed upstream of the NeoR expression cassette.
  • CreERT2 is a fusion protein containing a ligand binding region mutant (ERT2) of the estrogen receptor (ER) and Cre recombinase.
  • ERT2 ligand binding region mutant
  • ER estrogen receptor
  • Cre-ERT2 is in an inactive state in the cytoplasm.
  • 4-OHT estrogen analogue
  • ERT2 binds to ERT, allowing Cre-ERT2 to enter the nucleus and exert Cre recombinase activity.
  • the IRES-PuroR structure is used to screen positive clones for gene knock-in.
  • this application uses the FLP/FRT system to excise the vector sequence and G418 resistance expression cassette.
  • cells will express some unnecessary selective genes, such as HygR, NeoR and PuroR.
  • This application uses FLP/FRT recombination to delete these selective genes.
  • the NeoR-CreERT2-IRES-PuroR sequence can be deleted by the FRT sequence-dependent FLP recombinase.
  • this application can also use other resistance screening mechanisms known in the art. These commonly used resistance screening mechanisms are, for example, Puromycin, Hygromycin, HPRT, etc.
  • TAR-YAC-HygR- ⁇ NeoGFP- Loxp 66 FRT i.e. TAR-Loxp 66, see Figure 4 in the accompanying drawings of the specification
  • TAR-YAC-HygR- ⁇ NeoGFP-WT Loxp FRT i.e. TAR-WT Loxp
  • the vectors TAR-Loxp 66 and TAR-WT Loxp were obtained by DNA synthesis.
  • the TAR-Loxp 66 sequence (the full name is TAR-YAC-HygR- ⁇ NeoGFP-Loxp 66 FRT sequence) is SEQ ID NO: 25, see the sequence table.
  • the difference between the vector TAR-WT Loxp and TAR-Loxp 66 is that the Loxp 66 sequence (ATAACTTCGTATA ATGTATGC TATACGAACGGTA) of the TAR-Loxp 66 vector is replaced with the WT Loxp sequence (ATAACTTCGTATA ATGTATGC TATACGAAGTTAT).
  • the 340kb partial human IGL locus template sequence is derived from BAC vectors RP11-685C18, RP11-890G10 and RP11-373H24.
  • the above-mentioned BAC vector was digested by the Crispr/Cas9 method in vitro, and the above-mentioned linearized DNA fragment was cloned into the pTARYAC vector by the TAR cloning technology.
  • the recombinant pTARYAC vector can be amplified and purified in bacteria.
  • DNA fragment IGL1, 38kbp DNA fragment IGL2, about 155kb (GRCh37/hg19Chr22: 22415353- 22571119) and DNA fragment IGL3, 147kb (GRCh37/hg19 Chr22: 22570833-22718740).
  • homologous recombination sequence of about 100 bp between the above-mentioned digested DNA products.
  • the linearized TAR-WT Loxp and TAR-Loxp 66 were prepared by digestion with ApaI and PAC I and DNA gel purification. The ends of linearized TAR-WT Loxp and TAR-Loxp 66 contain the end homologous sequences of DNA fragment IGL1 and DNA fragment IGL3, respectively. Using the TAR cloning method, the digested DNA fragments IGL1, IGL2, IGL3 and linearized TAR-WT Loxp or TAR-Loxp 66 were transformed into Saccharomyces cerevisiae to obtain a circular vector containing 340kb human IGL locus that can be used for gene integration .
  • the yeast protoplast fusion method is abbreviated as follows:
  • the yeast cell culture was harvested overnight and then washed with 1M sorbitol. In the presence of 1M sorbitol, the cells were treated with ZymolyaseTM and ⁇ -mercaptoethanol to transform into spheroplasts. The spheroplasts were washed with sorbitol and resuspended in a buffer containing sorbitol and CaCl 2. Mix 200 ⁇ l of competent yeast equivalent to 5 ml of the original yeast culture with 100 ng of linearized vector and 100 ng of RP11-890G10 digested product. Incubate at 30°C for 30 minutes, then heat shock in a 42°C water bath for 20-25 minutes.
  • TAR-IGL-Loxp 66 TAR-IGL-HygR- ⁇ NeoGFP-WTLoxp
  • TAR-IGL-WTLoxp TAR-IGL-WTLoxp
  • this application uses this technology to insert expression cassettes containing CreERT2 and 5'NeoR at specific sites Chr16:19046551-19048556 (NCBI37/mm9) in embryonic stem cells (ES cells).
  • ES cells embryonic stem cells
  • guide RNA is designed near the insertion site (Chr16:19046551-19048556 (NCBI37/mm9)).
  • the recognition target sequence is TTGGCTACAATAGCCAATGC/CGG, and the CGG at the 3'end is the PAM sequence.
  • ES cells (EDJ#22) were purchased from ATCC (ATCC, cat: SCRC-1021) as an ES cell line derived from 129S5/SvEvTac.
  • the inserted gene structure is: Loxp71-hprt intron-5'Neo-PGK promoter-rBGpA-EF1a promoter-CreERT2-IRES-puro-FRT, and its sequence is shown in the sequence table.
  • the Loxp 71 sequence is used to mediate the site-directed integration of foreign genes.
  • Upstream of Loxp 71 is a truncated NeoR resistance expression cassette.
  • a complete NeoR expression cassette can be formed. This newly formed resistance expression cassette can be used to identify the site-directed insertion of foreign genes.
  • the CreERT2-IRES-PuroR structure was also designed upstream of the NeoR expression cassette.
  • CreERT2 is a fusion protein containing a ligand binding region mutant (ERT2) of the estrogen receptor (ER) and Cre recombinase.
  • ERT2 ligand binding region mutant
  • ER estrogen receptor
  • Cre-ERT2 is in an inactive state in the cytoplasm.
  • 4-OHT estrogen analogue
  • ERT2 a metabolite of Tamoxifen
  • the IRES-PuroR structure is used to screen positive clones for gene insertion.
  • the NeoR-CreERT2-IRES-PuroR sequence can be deleted by the FRT sequence-dependent FLP recombinase.
  • the 5'end portion of the Neo gene and the 3'end portion of the Neo are truncated at position 92, and the 5'end portion of the Neo gene expresses the amino acids from positions 1 to 91 of neomycin, and the Neo gene
  • the 3'end portion of neomycin expresses the amino acid from position 93 to 267
  • the 92nd amino acid R is the beginning of the 2 nucleotides AG at the end of the 5'end portion and the 3'end portion of the Neo gene.
  • the first nucleotide G is co-coded.
  • a 1014bp 5'recombination arm was cloned upstream of the insertion sequence (Loxp 71-5'NeoR-CreERT2-IRES-PuroR-FRT), and its sequence is located at chr16:19046411- 19047425 (NCBI37/mm9).
  • the 3'end recombination arm of 1087bp was cloned downstream, and its sequence is located at NCBI37/mm9 chr16:19047426-19048512 (NCBI37/mm9).
  • RNP Cas9 ribonucleoprotein complex
  • ES cells and 20ul RNP were mixed with DNA template, and electrotransfection was carried out in an electroporation cuvette (BioRad, cata:1652081). Electrotransfection conditions: 1200 volts, pulse interval 30 milliseconds. After electrotransfection, ES cells diluted with medium were cultured in a 10-cm culture dish with trophoblast cells (sigmaaldrich Cata: PMEF-CFX). The medium and culture conditions of ES cells refer to ATCC's instructions on this cell line.
  • ES 5'Neo-Loxp 71 The genetically modified ES clone was named ES 5'Neo-Loxp 71.
  • this application prepared another ES clone, the inserted gene structure is WT-Loxp-hprt intron-5'Neo-PGKpromoter-rBGpA-EF1a-CreERT2-IRES-puro-FRT.
  • the modified ES clone was named ES 5'Neo-WTLoxp.
  • the difficulty of directional insertion of large fragments of DNA into recipient cells lies in the use of traditional methods, such as liposome transfection, electroporation, etc.
  • the transformation efficiency is low, and the exogenous DNA in the form of linear large fragments is easily broken.
  • large fragments of DNA are purified from yeast cells. The efficiency is very low.
  • this application uses yeast spheroplast fusion technology. Under the mediation of PEG, yeast spheroplasts can fuse with cells to introduce large fragments of DNA into recipient cells.
  • yeast spheroplast-mediated cell fusion can be used to mediate the introduction of YAC containing large fragments of DNA into recipient cells
  • this introduction is mostly random chromosome insertion rather than site-specific insertion.
  • the linear YAC carrier is easily degraded or broken in the cell.
  • this application has made improvements. This application splices the YAC vector into a circular YAC vector, and at the same time loads large fragments of DNA into the circular YAC vector, thereby improving the efficiency of site-specific insertion of complete genes into cell chromosomes.
  • ES cells embryonic stem cells
  • the yeast solution loaded with large fragment IGL (carrying the TAR-IGL-Loxp 66 gene) was added to YPD (Sigma, Cata: Y1375) medium containing 0.2 mg/mL Hygromycin, and shaken overnight at 30°C. The next day, dilute the bacterial solution 10 times with YPD medium and continue to shake for 3-4 hours until the OD600 value is 2.0-3.0. Subsequently, it was centrifuged at 2500g for 15 minutes to collect the yeast. Suspend the yeast cell pellet with 20ml of 1M sorbitol and let it stand at 4°C for 2 hours.
  • Collect the yeast cells by centrifugation, mix the cell pellet with SPEM buffer (1M sorbitol, 0.1M sodium phosphate buffer mixture pH 7.4, and 10 mM EDTA, pH 8.0), then add 75ul 14mM 2-ME and 100UL Zymolase to remove the wall Treat and digest at 30°C for 30 minutes.
  • the digested cells were washed with 1M sorbitol.
  • the cells were suspended in 2ml of STC solution (1M sorbitol, 10mM Tris-HCl, pH 7.5, 10mM CaCl 2 and 2.5mM MgCl 2 ) for later use.
  • ES 5'Neo-Loxp 71 the above-mentioned genetically modified ES cells (for example, ES 5'Neo-Loxp 71) are collected.
  • ES 5'Neo-Loxp 71 cells and TAR-IGL-Loxp 66 yeast cells were mixed in a PBS solution at a ratio of 1:100. After centrifugation at room temperature, the cell mixture was suspended in a PEG1500 solution containing 10% DMSO (Roche Applied Science, cata: 10783641001). Two minutes later, the fused ES cells were suspended in the culture medium and centrifuged at 200g for 5 minutes. Suspend the cell mixture, dilute 2x10 6 ES cells with ES cell culture medium, and culture them in 5 10cm petri dishes.
  • Tamoxifen (1 ⁇ g/ml) (Sigma, Cata: T5648-1G) was added to induce ES cells to express Cre recombinase. After 16 hours, Tamoxifen was washed away, G418 was added, and resistant clones were screened.
  • ES 5'Neo-WTLoxp cells and TAR-IGL-WTLoxp yeast cells were fused by PEG, and then screened with G418, but no G418-resistant clones were observed, indicating that WT Loxp cannot mediate large fragment loops Or the insertion efficiency is very low.
  • this application also tried the conventional Cre-Lox recombination method, that is, using the wild-type Loxp sequence, but it failed to stably integrate the macromolecular DNA into the cell chromosomes. This may be because the Loxp sequence-dependent recombination reaction catalyzed by Cre is reversible, that is, reverse recombination or deletion may occur between Loxp sites, so the recombination between wild-type Loxp sites cannot effectively mediate the stability of macromolecular DNA Site-specific integration into cell chromosomes.
  • G418-resistant monoclonal ES cells (ES 5'Neo-WTLoxp) expressing GFP were picked and transferred to a 48-well culture plate to continue growth. After 3 days, use a pipette tip to disperse the ES cells, half of the cells are used to extract genomic DNA, and the other half are frozen in a 48-well cell culture plate for later use.
  • clones with G418 resistance will recombine between the Loxp 66 site of the foreign fragment and the Loxp 71 position of the chromosome, so that WTLoxp sites and Loxp 71/66 sites are generated at both ends of the insert. Design primers at both ends of WTLoxp and Loxp 71/66, and identify the ES clones inserted into the site by PCR.
  • the primer sequence is as follows:
  • WT Loxp F GGAGATCCTCAGGTCATTGC;
  • WT Loxp R GGCAGAGCTTTGCTTTTGTT;
  • Loxp 66/71 F CCTTGACCCAGAAATTCCAC;
  • Loxp 66/71 R TGGAGGCCATAAACAAGAAGA.
  • the primer design and its PCR identification results are shown in Figure 9 in the accompanying drawings of the specification.
  • the cell will express some unnecessary selective genes, such as HygR, NeoR, CreERT2 and PuroR. These selective genes can be deleted using FLP/FRT recombination.
  • Figure 3 in the accompanying drawings of the specification Synthesize FLP plasmid (pCAG-Flpe) sequence (Addgene, Cata: 13787).
  • the pCAG-Flpe plasmid was transformed into ES clones with IGL gene inserted, and GFP-negative, G418-sensitive and Puromycin-sensitive ES clones were selected for use.
  • Cre-Lox recombination is a site-specific recombinase technology used for deletion, insertion, transposition and inversion operations at specific sites in cell DNA.
  • the loxP site is composed of a 34bp special site sequence.
  • the middle 8bp DNA base is an asymmetric sequence, which determines the direction of the loxP sequence.
  • On both sides of the asymmetric sequence are two 13bp reverse symmetric sequences, which determine the binding efficiency with Cre.
  • Cre can still catalyze mutations between Loxp (such as Loxp 71 and Loxp 66). Recombination reaction. However, after the recombination reaction of Loxp 71 and Loxp 66, wild-type Loxp and double mutant Loxp (Loxp 71/66) were produced.
  • Loxp 71/66 has mutations in the symmetrical sequences at both ends, its ability to bind to Cre is greatly reduced, so the reverse recombination reaction between WT Loxp and Loxp 71/66 is avoided, thereby enhancing the stability of the inserted gene .
  • This application compares the above experiments and finds that this mutant Loxp, rather than the wild-type Loxp, facilitates the site-specific insertion of large fragments of DNA.
  • Loxp double mutation Loxp66/71 is the most preferred solution for mediating the stable and site-specific integration of macromolecular DNA into cell chromosomes.
  • Loxp with 1 mutation such as Loxp/Loxp66 or Loxp/Loxp71, or Loxp double mutation with other mutation positions
  • loxp66/71 has the best effect, the highest transformation efficiency, the most effective and stable Stable site-specific integration of macromolecular DNA into cell chromosomes, which is most suitable for site-specific integration and cloning of large fragments of DNA.
  • Primer Insert 5F used to screen the PCR of the genomic DNA integrated with the expression cassette:
  • the primer Insert 5R used to screen the PCR of the genomic DNA integrated with the expression cassette :
  • Primer Insert 3R for screening PCR of genomic DNA integrated with expression cassette

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Abstract

提供了一种向基因组中定点整合大片段外源DNA的方法，该方法使用了Cre/loxp系统，其中loxp是双突变loxp71/61，从而提高了整合的稳定性和效率。

Description

定点整合大片段外源DNA的方法 发明领域
本申请涉及基因工程技术领域。具体地，涉及定点整合目的基因片段的方法，本申请的方法适合将目的基因片段，尤其是300kb以上大片段外源DNA高效地定点整合到细胞的基因组中。
发明背景
对细胞的基因组进行大规模DNA修饰，尤其是对基因组中定点整合超长基因片段，尤其长度在300kb以上的外源基因大片段一直是生物技术的难点问题。
哺乳动物人工染色体(mammalian artificial chromosome，MAC)是从哺乳动物细胞中分离出的，并由复制起始区、端粒以及着丝粒构建而成的克隆载体。该载体可以用于装载大于1000kb的外源DNA片段。利用微细胞介导的染色体转移技术(microcell mediated chromosome transfer，MMCT)可以将MAC所携带的大片段外源基因导入真核细胞(Martella,Pollard et al.2016)。由于MAC是游离于染色体存在的，因此外源基因不能稳定地整合到细胞染色体内，并获得稳定的遗传。
酵母人工染色体(Yeast artificial chromosomes，YAC)是人工染色体中能克隆最大DNA片段的载体，其中可以插入100-2000kb的外源DNA片段。利用酵母原生质体融合(yeast spheroplast cell fusion)技术可以将装配有大片段外源DNA的YAC载体插入到受体细胞染色体中(Mendez,Green et al.1997)。但是这种办法存在一系列问题：外源基因拷贝数目不可控，基因组整合位置随机，YAC载体调控序列导致受体细胞受到基因污染，在细胞融合和整合过程中大片段基因容易断裂等。同时，利用这种方法建立转基因小鼠时，既需要对基因编辑的ES细胞克隆进行大量的基因型鉴定，也需要对不同的转基因小鼠系的表型进行鉴定。
细菌人工染色体(Bacterial artificial chromosome，BAC)是一种以F质粒 (F-plasmid)为基础构建而成的细菌染色体克隆载体，常用来克隆150kb-200kb大小的DNA片段。由于BAC载体具有容量大、遗传稳定、易于操作等优点，常被用来进行基因改造。利用传统的同源重组技术或重组酶系统可以将100kb到200kb的外源基因定点整合到胞基因组特定位置((Valenzuela,Murphy et al.2003；Wallace,Marques-Kranc et al.2007)。受到BAC载体基因容量的限制，如果希望对基因组进行更大规模的修饰，如定点插入或原位替换，则需要对细胞进行复杂的多次基因修饰(Macdonald,Karow et al.2014；Murphy,Macdonald et al.2014)。这种策略无疑周期长，技术难度高。
因此，本领域急需对基因组中定点整合大片段外源DNA，尤其300kb以上大片段外源DNA的方法。
发明概述
对细胞的基因组进行大规模DNA修饰，特别是将大片段的外源DNA定点整合到基因组中，一直是当前生物技术难点问题。定点整合的主要技术难点包括如下方面：1).难以将大片段的外源DNA克隆到基因载体里；2).难以完整地将含有大片段外源DNA的载体导入真核细胞；3).难以高效地将大片段外源DNA定点整合到受体细胞的基因组中；4).难以高效地筛选并检测定点整合重组细胞克隆。
本专利通过一系列技术革新，解决了上述技术难题，首次提供了一种对基因组中高效稳定地定点整合大片段外源DNA，尤其300kb以上大片段外源DNA的方法。
具体来说，一方面本申请利用了改良的重组酶系统Cre/Loxp，即利用Loxp71/66突变体将外源DNA稳定地不可逆地定点整合到受体细胞的基因组中。
Cre-Lox重组是一种用于在细胞DNA的特定位点上进行删除、插入、转座和倒位操作的位点特异性重组酶技术。Loxp位点由34bp的特殊位点序列组成，其中中间8bpDNA碱基是不对称序列，决定了Loxp序列的方向，不对称序列两边是两段13bp反向对称序列，决定了与Cre的结合效率。
虽然常规的Cre-Lox重组方法可催化外源基因的定点整合。但是在最初的实验中，野生型的Loxp不能将大分子DNA稳定地定点整合到细胞染色体。经分析，这可能是因为Cre催化的Loxp序列依赖的重组反应是可逆的，即Loxp位点之间可能发生反向重组或删除，所以野生型Loxp位点不能有效地介导大分子DNA稳定地定点整合到细胞染色体。
当对称序列的一端发生突变，如Loxp71(5’端对称序列存在突变)和Loxp66(3’端对称序列存在突变),Cre仍然可以催化突变Loxp之间(如Loxp71和Loxp66)发生重组反应。Loxp71和Loxp66发生重组反应后，产生野生型Loxp和双突变Loxp(Loxp71/66)。因为Loxp71/66的两端的对称序列都发生突变，其和Cre的结合能力大为降低，因此避免了野生型Loxp和Loxp71/66之间发生反向重组反应，导致外源DNA稳定地不可逆地整合到细胞染色体里，从而增强了敲入基因的稳定性。本申请首次发现了，这种突变型Loxp，而非野生型Loxp，有助于大片段DNA的定点整合到细胞基因组中。原理如说明书附图中的图2。
本申请通过实验发现，对于介导大分子DNA稳定地定点整合到细胞染色体，Loxp双突变Loxp66/71是最优选的方案。相对于野生型Loxp，或者含1个突变的Loxp，例如Loxp/Loxp66或者Loxp/Loxp71，或者含其他突变位置的Loxp双突变，loxp66/71的效果最好，转化效率最高，能最有效最稳定地将大分子DNA稳定地定点整合到细胞染色体，最适合于大片段DNA的定点整合和克隆。
另外，为了高效地筛选定点整合到染色体上的重组子，本申请还分别在DNA和染色体的Loxp位点之后设计了功能互补且截断型的抗性基因新霉素(NeoR)的表达盒。只有定点位置插入外源基因的重组子才可以表达G418药物的抗性，随机整合的DNA片段则不能产生药物抗性。示意图见说明书附图中的图3。
同时，本申请使用了FLP/FRT系统来切除不必要的载体序列和G418抗性表达盒。基因修饰元件及其重组原理见说明书附图中的图3。
此外，由于YAC载体最多可以容纳2000kb的外源DNA片段，本申请选用YAC作为装载外源基因的载体。传统的YAC载体是线性结构，在细胞内不稳 定，容易发生断裂和同源重组。对此，本申请利用YAC载体将大片段DNA装载到环状的YAC载体中，从而提高了完整基因定点插入细胞染色体的效率。
大片段DNA定向敲入受体细胞的难度在于转化效率低，且外源DNA容易断裂，而且大片段DNA从酵母细胞纯化效率很低，因此本申请选用了酵母原生质球融合技术(yeast protoplast fusion)。将酵母内的大片段外源基因导入到受体细胞。这种方法对外源基因体大小没有限制，而且可以避免外源基因在纯化过程中发生断裂(Brown，Chan et al.2017)。
本专利通过以上一系列的技术设计从而克服了大分子DNA定点整合染色体DNA过程中存在的各种问题，如大分子DNA转染效率差，外源基因易断裂，定点整合效率差等。因此本申请的方法可以高效稳定地将外源DNA，尤其是大片段外源DNA，更特别地300kb以上大片段外源DNA一次性地定点整合到基因组中。
具体地，本发明涉及以下方面：
1.整合目的基因片段的方法，其包括以下步骤：
1)获得目的基因片段；
2)将目的基因片段克隆到载体中，在所获得的包含目的基因片段的载体中插入了抗性表达盒1，优选截断型新霉素(G418)抗性表达盒1，所述表达盒1在5’至3’方向上包含突变型Loxp 1，优选Loxp66序列、部分HPRT内含子和抗性基因a部分，优选新霉素抗性基因Neo基因3’端部分；
3)将含有所述目的基因片段的载体转化到微生物细胞内，优选为酵母，尤其是酿酒酵母细胞内；
4)将所述微生物内含有目的基因片段的所述载体导入到受体细胞，其中所述受体细胞的基因组中已经定点导入了抗性表达盒2，优选截断型新霉素(G418)抗性表达盒2，所述表达盒2在5’至3’方向上包含部分HPRT内含子、突变型Loxp 2，优选Loxp71序列、抗性基因b部分，优选Neo基因5’端部分、含有雌激素受体(estrogen receptor，ER)的配体结合区突变体(ERT2)与Cre重组酶的融合蛋白CreERT2，和IRES-PuroR结构；
其中所述突变型Loxp 1和突变型Loxp 2中的突变分别位于野生型Loxp位点的3’对称序列和5’对称序列，或者野生型Loxp位点的5’对称序列和3’对称 序列，所述突变型Loxp 1和突变型Loxp 2均能与Cre结合，在所述步骤4)后形成两个末端都含有突变的Loxp序列，如Loxp66/71；
优选地，单端突变的loxp序列，突变型Loxp 1和突变型Loxp 2，例如Loxp71和Loxp66不影响与Cre的结合效率，而在重组后形成两个loxp序列，其中一个是野生型Loxp序列，另一个是两个末端都含有突变的Loxp序列，如Loxp66/71，后者相比于野生型Loxp序列降低了与Cre的结合效率；
现有技术中已知类似于Loxp66序列，有可能与loxp71重组形成双末端突变Loxp结构包括loxJTZ17、loxKR1、loxKR2、loxKR3、loxKR4等。这些类似的Loxp结构在不同细胞中介导重组，整合到染色体形成稳定重组子的效率略有不同。并且，整合到染色体形成稳定重组子的效率受多种因素的影响，例如所整合的细胞系。本申请的Loxp66/71结构相对于这些类似的Loxp突变体在细胞的染色体中形成稳定重组子的效率最高，因此DNA大片段克隆效率最高；
5)诱导CreERT2的重组酶活性，优选通过加入Tamoxifen诱导CreERT2的重组酶活性，所述Cre重组酶介导所述目的基因片段定点整合到所述受体细胞的基因组中，同时所述表达盒1和表达盒2形成表达完整抗性基因，优选新霉素的抗性表达盒；
6)筛选基因组中定点整合了所述目的基因片段的所述受体细胞。
2.根据项1的方法，其中在所述步骤2)中的抗性表达盒1中所包含的抗性基因a部分，优选新霉素抗性基因Neo的3’端部分和所述步骤4)中的抗性表达盒2中的抗性基因b部分，优选Neo基因5’端部分是功能互补的，任一单独的抗性表达盒1或2均不能使细胞产生抗性，例如截断型新霉素(G418)抗性表达盒1或2均不能使细胞产生新霉素抗性，优选地只有其中包含的抗性基因b部分和a部分，例如Neo基因5’端部分和3’端部分按照一定顺序重组之后才可以表达有新霉素抗药性蛋白。
优选地，所述Neo基因5’端部分和所述Neo的3’端部分是在92位截断的，更优选地，新霉素的第92位氨基酸是由所述Neo基因5’端部分的末尾核苷酸和Neo基因的3’端部分的起始核苷酸共同编码的，更优选地，新霉素的第92位氨基酸是由所述Neo基因5’端部分的末尾2个核苷酸和Neo基因的3’端部分 的起始处第一个核苷酸共同编码的。也就是说，Neo基因的5’端部分表达新霉素从第1至91位的氨基酸，Neo基因的3’端部分表达新霉素从第93位到267位的氨基酸，同时5’端部分的末尾2个核苷酸和Neo基因的3’端部分的起始处第一个核苷酸共同编码新霉素的第92位氨基酸，
并且所述步骤6)通过加入特异性针对所述抗性基因的试剂，优选抗生素G418来筛选基因组中定点整合了所述目的基因片段的所述受体细胞。
3.根据项1或2的方法，其中在所述步骤1)中的所获得的包含目的基因片段的载体中，所述目的基因片段的上游存在FRT序列，同时在所述步骤4)中的表达盒中，在所述IRES-PuroR结构的下游存在FRT序列，
并且在所述步骤6)之后还包括步骤7)：将包含FLP的质粒转入所述受体细胞以除去所述表达盒。
4.根据前述项中之一的方法，其中所述步骤1)中的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。
5.根据前述项中之一的方法，其中所述步骤2)中所获得的包含目的基因片段的载体是环状BAC载体或基因组DNA。
6.根据前述项中之一的方法，其中所述步骤2)中的所述载体是线性化载体，所述线性化载体优选为拥有被克隆目的DNA片段同源末端序列的线性化载体，更优选为线性化酵母穿梭载体。
7.根据项6的方法，其中所述线性化载体是YAC或TAR克隆载体。
8.根据项7的方法，其中所述线性化载体是YAC克隆载体，优选pTARYAC克隆载体。
9.根据项1-5中之一的方法，其中所述步骤2)中的所述包含目的基因片段的载体是BAC(bacterial artificial chromosome)或PAC(P1 artificial chromosomes)。
10.根据项5的方法，其中所述环状载体是通过同源重组拼接技术(优选酵母的转化偶联重组(TAR)技术)，或者同源重组拼接技术(优选酵母的转化偶联重组(TAR)技术)与基因组编辑技术的组合获得的。
11.根据项10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)，酵母的转化偶联重组(TAR)技术和细菌的Red/ET同源重组技术，优选酵母的转化偶联重组(TAR)技术。
12.根据前述项中之一的方法，其中所述步骤1)中的目的基因片段是从染色体或另外的载体(优选BAC载体)上获得的。
13.根据前述项中之一的方法，其中所述步骤1)中的目的基因片段是利用基因组编辑技术对所述染色体或载体进行切割获得的。
14.根据前述项中之一的方法，其中在所述步骤4)中通过基因组编辑技术将所述表达盒及其下游大片段DNA定点稳定地导入到所述受体细胞的基因组中。
15.根据项10、13或14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。
16.根据前述项中之一的方法，其中所述酵母是酿酒酵母(saccharomyces cerevisiae)。
17.根据前述项中之一的方法，其中在所述步骤4)中使用酵母原生质球融合(yeast protoplast fusion)技术将将所述微生物内含有目的基因片段的所述载体导入到受体细胞。
18.根据前述项中之一的方法，其中所述步骤2)中的抗性表达盒1，优选截断型新霉素(G418)抗性表达盒1中的所述突变型Loxp 1和/或所述步骤4)中的抗性表达盒2，优选截断型新霉素(G418)抗性表达盒2中所述突变型Loxp 2用于将所述表达盒定点导入所述受体细胞的基因组中。
19.根据前述项中之一的方法，其中所述步骤2)中的抗性表达盒1，优选截断型新霉素(G418)抗性表达盒1中包含的HPRT内含子、抗性基因a部分，优选Neo基因3’端部分和所述步骤4)中的抗性表达盒2，优选截断型新霉素(G418)抗性表达盒2中包含的HPRT内含子、抗性基因b部分，优选Neo基因 5’端部分，以及步骤5)中形成的表达完整抗性基因的抗性表达盒，优选新霉素Neo的新霉素(G418)抗性表达盒，用于筛选目的基因片段定点整合后的细胞重组子。
20.根据前述项中之一的方法，其中所述步骤4)中的受体细胞是真核细胞，优选动物胚胎干细胞。
另外，除了利用互补截断的G418基因抗性来筛选定点位置插入了外源基因的重组子之外，本申请也可以使用本领域已知的其他抗性筛选机制。这些常用的抗性筛选机制或者抗性基因可以为例如为嘌呤霉素(Puromycin)、潮霉素(Hygromycin)、HPRT等。
附图简述
图1.显示了大片段外源基因定点整合到细胞基因组中的总体策略。
图2.Loxp71/66突变体促进大分子环状DNA的定向整合。
图3.外源环状DNA定点整合细胞染色体示意图。
图4.TAR-YAC-HygR-ΔNeoGFP-Loxp66 FRT质粒图谱。
图5.载体TAR-IGL-Loxp66 PCR鉴定电泳结果。
在图5中，
M表示100bp DNA ladder，
泳道1是载体TAR-Loxp66末端和IGL1连接处PCR产物1，
泳道2是IGL1内部PCR产物2，
泳道3是IGL1和IGL2的连接处PCR产物3，
泳道4是IGL2内部PCR产物4，
泳道5是IGL2和IGL3连接处PCR产物5，
泳道6是IGL3内部PCR产物6，和
泳道7是IGL3和载体TAR-Loxp66末端连接处PCR产物7。
图6.用于鉴定TAR-IGL-Loxp66的引物序列。
图7.CRISPR/Cas9技术介导的基因敲入。
图8.鉴定基因修饰的受体细胞。
图9.鉴定大片段IGL的定点插入。
图10.截断型新霉素(G418)抗性表达盒2中的Neo基因5’端部分和截断型新霉素(G418)抗性表达盒1中所包含的新霉素抗性基因Neo的3’端部分的核苷酸序列和氨基酸序列示意图。图中所述Neo基因5’端部分和所述Neo的3’端部分是在92位截断的，Neo基因的5’端部分表达新霉素从第1至91位的氨基酸，Neo基因的3’端部分表达新霉素从第93位到267位的氨基酸，同时5’端部分的末尾2个核苷酸和Neo基因的3’端部分的起始处第一个核苷酸共同编码新霉素的第92位氨基酸R。图10中，上端未加阴影部分显示的是Neo基因的5’端部分表达新霉素从第1至91位的氨基酸序列极其相对应的核苷酸序列，下端加灰色阴影部分显示的是Neo基因的3’端部分表达新霉素从第93位到267位的氨基酸序列极其相对应的核苷酸序列。同时，加黑色阴影部分显示的是第92位氨基酸R，是由5’端部分的末尾2个核苷酸AG和Neo基因的3’端部分的起始处第一个核苷酸G共同编码的。
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。参见例如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 Press1991)；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，现有技术中缺少对基因组中定点整合针对300kb以上的大片段基因组DNA的方法。
为了解决此问题，本发明建立了一种高效稳定地定点整合300kb以上基因，特别是真核基因到基因组中的方法。与现有技术相比，本申请的方法不仅能够将大的外源DNA片段，例如300kb以上稳定地整合到基因组中，同时还提高了定点整合的效率。
如上所述，对细胞的基因组进行大规模DNA修饰，特别是将大片段的外 源DNA定点整合到基因组中，一直是当前生物技术难点问题。定点整合的主要技术难点包括如下方面：1.如何将大片段的外源DNA克隆到基因载体里；2.如何完整地将含有外源DNA的载体导入真核细胞；3.如何高效地将大片段外源DNA定点整合到受体细胞的基因组中；4.如何高效地筛选并检测定点整合重组细胞克隆。
对于上述4个技术难点，本申请均进行了设计从而能够高效稳定地将外源DNA定点整合到基因组中。具体如下：
对于1)：如何将大片段的外源DNA克隆到基因载体里
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的外源DNA在细胞内重组。同时，考虑到YAC载体最多可以容纳2000kb的外源DNA片段，本申请选用YAC作为装载外源基因的载体。传统的YAC载体是线性结构，在细胞内不稳定，容易发生断裂和随机重组。对此，本申请利用YAC载体将大片段DNA装载到环状的YAC载体里，从而提高了完整基因定点插入细胞染色体的效率。
在一个实施方案中，利用同源重组拼接技术，或者同源重组拼接技术与基因组编辑技术的组合获得含有目的基因片段的环状YAC载体。在一个优选的实施方案中，使用同源重组拼接技术，例如酵母转化偶联重组(Transformation-associated recombination,TAR)技术将线型的YAC载体环化为环状闭合的YAC载体。在另一个优选的实施方案中，联合使用同源重组拼接技术与基因组编辑技术，例如Crispr/Cas9方法和TAR技术将多个BAC载体里的基因片段编辑成多个有同源末端的线性DNA段，并利用酿酒酵母的同源重组特性，在其细胞内拼接成环状的YAC载体。具体方法可参见中国专利申请号201811238618.6。
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)，酵母的转化偶联重组(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(P1 artificial  chromosomes)。
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)。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质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，本申请基于酵母特有的高效DNA同源重组机制，在酵母细胞内利用YAC载体将大片段DNA装载到环状的YAC载体里，从而提高了完整基因定点插入细胞染色体的效率。
对于2)：如何完整地将含有大片段外源基因的载体导入真核细胞
大片段DNA定向敲入受体细胞的难度在于转化效率低，且外源DNA容易断裂，而且在体外从酵母细胞纯化大片段DNA的效率很低，同时普通DNA转染方法对DNA的大小也有限制。
对此，作为改进，本申请采用了酵母原生质球融合(yeast protoplast fusion)的方法将酵母内的大片段外源基因导入到受体细胞。在PEG的介导下,酵母原生质球可以和细胞融合，大片段DNA被导入受体细胞。这种方法对外源基因体大小没有限制，而且可以避免外源基因在纯化过程中发生断裂(Brown,Chan et al.2017)。
大片段外源基因转化及定点整合到细胞基因组中的过程参见说明书附图中的图1。
对于3)如何高效地将大片段外源基因组DNA定点整合到受体细胞的基因组中；
本申请利用了改良的重组酶系统Cre/Loxp，即利用Loxp71/66突变体将外源DNA稳定不可逆地定点整合到受体细胞的基因组中。
利用传统的重组酶系统,如Cre/Loxp,FLP/FRT和phiC31-att等，虽然可以将环状的载体定点插入到细胞染色体中(Ohtsuka,Miura et al.2012)。但是尚未有上述重组酶可将超过300kb的外源环状基因一次性定点整合到细胞基因组中的报道。由于Cre/Loxp,FLP/FRT系统介导可逆的酶促反应，当大分子环状DNA重组到染色体时，重组酶介导的DNA切割反应远大于定点整合反应。这导致外源分子越大，获得的稳定的重组子效率越低。虽然phiC31-att重组系统可以介导DNA片段之间发生不可逆的重组反应，但是phiC31活性比Cre重组酶活性低，因此也并非介导大分子DNA重组的优选方案。在上述重组酶系统系统中， Cre/Loxp系统催化效率最高，使用最普遍，因此本申请考虑使用Cre/Loxp系统来介导大片段DNA的重组。
Cre-Lox重组是一种用于在细胞DNA的特定位点上进行删除、插入、转座和倒位操作的位点特异性重组酶技术。Loxp位点由34bp的特殊序列组成，其中中间8bpDNA碱基是不对称序列，决定了Loxp序列的方向，不对称序列两边是两段13bp反向对称序列，决定了与Cre的结合效率。
虽然常规Cre-Lox重组可催化外源基因的定点整合。但在最初试验中发现野生型的Loxp序列没能将大分子DNA稳定地定点整合到细胞染色体。这可能是因为Cre催化的Loxp序列依赖的重组反应是可逆的，即Loxp位点之间可能发生反向重组或删除，因此野生型Loxp位点之间的重组不能有效地介导大分子DNA稳定地定点整合到细胞染色体。
虽然在DNA片段两侧位点引入异源的Loxp序列(如Loxp和Loxp2272)可以介导同源Loxp位点间(Loxp和Loxp,Loxp2722和Loxp2722)发生重组，从而介导外源DNA定向插入染色体。但是Cre-lox重组的效率随着其插入长度的增大而大为降低。这种方案也许可以用于某些大分子DNA的定点插入，但也并非优选方案。
对此，在一个实施方案中，本申请利用tamoxifen诱导受体细胞，例如ES细胞表达Cre重组酶。当Loxp序列中的对称序列的一端发生突变，即突变型Loxp 1，优选Loxp71(5’端对称序列存在突变)和突变型Loxp 2，优选Loxp66(3’端对称序列存在突变)。
同时，其中所述突变型Loxp 1和突变型Loxp 2中的突变分别位于野生型Loxp位点的3’对称序列和5’对称序列，或者野生型Loxp位点的5’对称序列和3’对称序列，所述突变型Loxp 1和突变型Loxp 2均能与Cre结合，在所述步骤4)后形成两个末端都含有突变的Loxp序列，如Loxp66/71；
优选地，单端突变的loxp序列，突变型Loxp 1和突变型Loxp 2，例如Loxp71和Loxp66不影响与Cre的结合效率，而在重组后形成两个loxp序列，其中一个是野生型Loxp序列，另一个是两个末端都含有突变的Loxp序列，如Loxp66/71，后者相比于野生型Loxp序列降低了与Cre的结合效率；
现有技术中已知类似于Loxp66序列，有可能与loxp71重组形成双末端突 变Loxp结构包括loxJTZ17、loxKR1、loxKR2、loxKR3、loxKR4等。这些类似的Loxp结构在不同细胞中介导重组，形成稳定整合到染色体形成稳定重组子的效率略有不同。并且，整合到染色体形成稳定重组子的效率受多种因素的影响，例如所整合的细胞系。本申请的Loxp66/71结构相对于这些类似的Loxp突变体在细胞的染色体中形成稳定重组子的效率最高，因此DNA大片段克隆效率最高。
在一个优选的实施方案中，单端突变的loxp序列，突变型Loxp 1和突变型Loxp 2，优选Loxp71和Loxp66不影响与Cre的结合效率，而在重组后形成两个loxp序列，其中一个是野生型Loxp序列，另一个是两个末端都含有突变的Loxp序列，优选Loxp66/71，后者相比于野生型Loxp序列降低了与Cre的结合。
重组后环状DNA可以定点插入染色体并在插入片段两侧产生野生型的Loxp位点和Loxp 1/2型突变体，例如Loxp71/66突变体。因为Loxp 1/2型突变体，优选Loxp71/66突变体的两端的对称序列都发生突变，其和Cre的结合能力大为降低，因此Cre不能有效地催化Loxp 1/2型突变体，优选Loxp71/66突变体发生反向重组，从而导致大片段的环状DNA可稳定不可逆地整合到细胞染色体里。也就是说，Loxp 1/2型突变体，优选Loxp71/66突变体避免了野生型Loxp和Loxp1/2型突变体，优选Loxp71/66突变体之间发生反向重组反应，导致外源DNA稳定地不可逆地整合到细胞染色体里，从而增强了插入基因的稳定性。
如本申请实施例中显示的，野生型Loxp位点不能有效地通过Cre介导大型环状DNA稳定地定点整合到染色体。Loxp71/66突变体促进大分子环状DNA的定向整合工作原理参见说明书附图中的图2。
因此本申请首次发现了，突变型Loxp，例如Loxp71/66突变体而非野生型Loxp，有助于高效稳定地将大片段DNA定点整合到细胞基因组中。
同时，本申请通过实验发现，对于介导大分子DNA稳定地定点整合到细胞染色体，Loxp双突变Loxp66/71是最优选的方案。相对于野生型Loxp，或者含1个突变的Loxp，例如Loxp/Loxp66或者Loxp/Loxp71，或者含其他突变位置的Loxp双突变，loxp66/71的效果最好，转化效率最高，能最有效最稳定地将大分子DNA稳定地定点整合到细胞染色体，最适合于大片段DNA的定点整合和克隆。
对于4)如何高效地筛选并检测定点整合重组细胞克隆。
一旦目的基因片段定点整合到受体细胞的基因组中之后，还需要对受体细胞进行筛选。对此，载体一般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。
对于本申请，为了高效地筛选定点整合到染色体上的重组子，本申请还分别在DNA和染色体的Loxp位点之后设计了功能互补且截断型的抗性基因新霉素(NeoR)的表达盒。只有定点位置插入外源基因的重组子才可以表达G418药物的抗性，随机整合的DNA片段则不能产生药物抗性。因此，这个表达盒可以用于鉴定外源基因的定点插入。任一单独的截断型新霉素(G418)抗性表达盒均不能使细胞产生新霉素抗性，优选地只有其中包含的Neo基因的5’端部分和3’端部分按照一定顺序重组之后才可以表达有新霉素抗药性蛋白。
在一个优选的实施方案中，所述Neo基因5’端部分和所述Neo的3’端部分是在92位截断的。更优选地，新霉素的第92位氨基酸是由所述Neo基因5’端部分的末尾核苷酸和Neo基因的3’端部分的起始核苷酸共同编码的。在一个具体的实施方案中，新霉素的第92位氨基酸是由所述Neo基因5’端部分的末尾2个核苷酸和Neo基因的3’端部分的起始处第一个核苷酸共同编码的。也就是说，Neo基因的5’端部分表达新霉素从第1至91位的氨基酸，Neo基因的3’端部分表达新霉素从第93位到267位的氨基酸，同时5’端部分的末尾2个核苷酸和Neo基因的3’端部分的起始处第一个核苷酸共同编码新霉素的第92位氨基酸。
同时，在NeoR表达盒的上游还设计了CreERT2-IRES-PuroR结构。CreERT2是含有雌激素受体(estrogen receptor，ER)的配体结合区突变体(ERT2)与Cre重组酶的融合蛋白。在无Tamoxifen诱导的情况下，Cre-ERT2在细胞质内 处于无活性状态。当Tamoxifen诱导后，Tamoxifen的代谢产物4-OHT(雌激素类似物)与ERT结合，可使Cre-ERT2进细胞核发挥Cre重组酶活性。IRES-PuroR结构用于筛选基因敲入的阳性克隆。
另外，本申请使用了FLP/FRT系统来切除载体序列和G418抗性表达盒。随着外源基因插入到染色体基因组中，细胞会表达一些不必要的选择性基因，如HygR、NeoR和PuroR等。本申请利用FLP/FRT重组删除这些选择性基因。当外源基因整合到受体细胞染色体的Loxp71位点之后，NeoR-CreERT2-IRES-PuroR序列可以被FRT序列依赖的FLP重组酶删除。
另外，除了利用互补截断的G418基因抗性来筛选定点位置插入了外源基因的重组子之外，本申请也可以使用本领域已知的其他抗性筛选机制。这些常用的抗性筛选机制例如为嘌呤霉素(Puromycin)、潮霉素(Hygromycin)、HPRT等。
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:将部分人Lamda轻链基因座(IGL)定点整合到胚胎干细胞(ES细胞)中
1.制备装大片段外源DNA的YAC载体
根据以前报道的DNA大分子克隆方法(中国专利申请号201811238618.6),将340kb部分人IGL基因座(GRCh37/hg19 chr22:22,377,297-22,717,584)分别克隆到线形TAR克隆穿梭载体TAR-YAC-HygR-ΔNeoGFP-Loxp 66 FRT(即TAR-Loxp 66，参见说明书附图中的图4)和TAR-YAC-HygR-ΔNeoGFP-WT Loxp FRT(即TAR-WT Loxp)。通过DNA合成获得载体TAR-Loxp 66和TAR-WT Loxp。TAR-Loxp 66序列(全称为TAR-YAC-HygR-ΔNeoGFP-Loxp 66 FRT序列)为SEQ ID NO:25，见序列表。载体TAR-WT Loxp和TAR-Loxp 66的区别是，TAR-Loxp 66载体的Loxp 66序列(ATAACTTCGTATA  ATGTATGC TATACGAACGGTA)，被替换为WT Loxp序列(ATAACTTCGTATA ATGTATGC TATACGAAGTTAT)。
340kb的部分人IGL基因座的模板序列源自BAC载体RP11-685C18,RP11-890G10和RP11-373H24。按照专利方法(中国专利申请号201811238618.6)，利用Crispr/Cas9方法体外酶切上述BAC载体，再利用TAR克隆技术将上述线形化的DNA片段克隆到pTARYAC载体里。重组后的pTARYAC载体可在细菌内扩增并纯化。经I-SceI酶切大量纯化后的载体，得到3个DNA片段，分别是DNA片段IGL1，38kbp(GRCh37/hg19 Chr22:22377208-22415482)；DNA片段IGL2,约155kb(GRCh37/hg19 Chr22：22415353-22571119)和DNA片段IGL3，147kb(GRCh37/hg19 Chr22：22570833-22718740)。上述酶切DNA产物之间有约100bp左右的同源重组序列。
利用ApaI和PAC I酶切和DNA胶纯化制备线性化的TAR-WT Loxp和TAR-Loxp 66。线性化TAR-WT Loxp和TAR-Loxp 66的末端分别含有DNA片段IGL1和DNA片段IGL3的末端同源序列。利用TAR克隆方法将经酶切的DNA片段IGL1，IGL2，IGL3和线性化的TAR-WT Loxp或TAR-Loxp 66转化到酿酒酵母，得到可用于基因整合的含有340kb人IGL基因座的环状载体。
具体试验操作如下:
参照专利申请(中国专利申请号201811238618.6)所列方法，将100ng DNA片段IGL1((GRCh37/hg19 Chr22:22377208-22415482)，100ngDNA片段IGL2(GRCh37/hg19 Chr22：22415353-22571119)，100ngDNA片段IGL3(GRCh37/hg19 Chr22：22570833-22718740)分别和100ng的线性化的载体TAR-WT Loxp以及TAR-Loxp 66混合，使用酵母原生质融合方法(Kouprina and Larionov 2008)将DNA片段转化到感受态酵母细胞AB 1380(ATCC,Cata：20843)。
酵母原生质融合方法简略如下：
过夜收获酵母细胞培养物，然后用1M山梨醇洗涤。在1M山梨醇的存在下， 用ZymolyaseTM和β-巯基乙醇处理细胞转变为原生质球。用山梨醇洗涤所述原生质球，并重悬于含山梨醇和CaCl 2的缓冲液中。将200μl的等同于5ml原始酵母培养物的感受态酵母与100ng线性化载体，100ng RP11-890G10酶切产物混合。30℃温育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。将1.0ml YPD液体培养基(Sigma Cata#Y1375)加到每个反应管中，用微量移液器轻轻悬浮沉淀细胞，并在30℃培养过夜。
第二天，将等分的200μl转化混合液均匀涂抹在含有0.2mg/mL Hygromycin B的YPD固体培养基上以对其进行培养。48小时后，挑取阳性克隆并进行扩增，使阳性克隆在含有Hygromycin B的YPD液体培养基生长。使用试剂盒(
Figure PCTCN2020118294-appb-000001
Yeast DNA Kit，D3370-01)提取酵母DNA，利用PCR检测鉴定阳性克隆。使用不同的PCR引物对完整的重组子进行鉴定。最终得到两个重组子，分别是TAR-IGL-HygR-ΔNeoGFP-Loxp 66(即TAR-IGL-Loxp 66)和TAR-IGL-HygR-ΔNeoGFP-WTLoxp(即TAR-IGL-WTLoxp)。连接产物，即载体TAR-IGL-Loxp 66 PCR的电泳鉴定结果参见说明书附图中的图5。引物序列参见说明书附图中的图6。
2.受体细胞的基因修饰
为了将外源基因定点整合到宿主细胞染色体特定位置，需要对宿主细胞的染色体进行基因修饰。
考虑到CRISPR/Cas9技术周期短、效率高的优点，本申请利用该技术在胚胎干细胞(ES细胞)特定位点Chr16:19046551-19048556(NCBI37/mm9)插入含有CreERT2和5'NeoR的表达盒。工作原理见说明书附图中的图7。
具体来说，在插入位点(Chr16:19046551-19048556(NCBI37/mm9))位点附近设计引导RNA(Guide RNA)。其识别靶序列为TTGGCTACAATAGCCAATGC/CGG，其中3’端的CGG为PAM序列。ES细胞(EDJ#22)购自ATCC(ATCC,cat:SCRC-1021)为129S5/SvEvTac来源ES细胞系。
被插入基因结构为:Loxp71-hprt intron-5'Neo-PGK promoter-rBGpA-EF1a promoter-CreERT2-IRES-puro-FRT，其序列见序列表。其中Loxp 71序列用于介导外源基因的定点整合。在Loxp 71位点上游是截断的NeoR抗性表达盒。当外源基因插入到Loxp 71位点之后，可以形成完整的NeoR表达盒。这个新形成的抗性表达盒可以用于鉴定外源基因的定点插入。
在NeoR表达盒的上游还设计了CreERT2-IRES-PuroR结构。CreERT2是含有雌激素受体(estrogen receptor，ER)的配体结合区突变体(ERT2)与Cre重组酶的融合蛋白。在无Tamoxifen诱导的情况下，Cre-ERT2在细胞质内处于无活性状态。当Tamoxifen诱导后，Tamoxifen的代谢产物4-OHT(雌激素类似物)与ERT结合，可使Cre-ERT2进细胞核发挥Cre重组酶活性。IRES-PuroR结构用于筛选基因插入的阳性克隆。当外源基因整合到染色`体的Loxp 71位点之后，NeoR-CreERT2-IRES-PuroR序列可以被FRT序列依赖的FLP重组酶删除。
在本实施例中，Neo基因5’端部分和所述Neo的3’端部分是在92位截断的，Neo基因的5’端部分表达新霉素从第1至91位的氨基酸，Neo基因的3’端部分表达新霉素从第93位到267位的氨基酸，同时第92位氨基酸R是由5’端部分的末尾2个核苷酸AG和Neo基因的3’端部分的起始处第一个核苷酸G共同编码的。
为将上述基因结构定点插入宿主细胞的基因组，在插入序列(Loxp 71-5’NeoR-CreERT2-IRES-PuroR-FRT)的上游克隆了1014bp的5’端重组臂，其序列位于chr16:19046411-19047425(NCBI37/mm9)。在其下游克隆了1087bp的3’端重组臂，其序列位于NCBI37/mm9 chr16:19047426-19048512(NCBI37/mm9)。
具体试验方法如下:
A.制备Cas9 ribonucleoprotein复合体(RNP)：根据模板序列(ttggctacaatagccaatgc/cgg)利用化学合成的方法合成该序列的CrRNA(IDTdna公司)。CrRNA除了包含20nt的靶位点的特异序列，还包含16nt用于融合tracrRNA的互补序列。将10μM CrRNA和10μM的TracrRNA(IDTdna  cata:1072534)在95℃变性5min。随后冷却室温。加入等摩尔的Cas9 Nuclease V3,(IDTdna cata:1081058)，以PBS作为缓冲液，反应溶液体积为20ul，在25℃温育20min形成RNP。
B.使20μl RNP和100ng线性的模板DNA(Loxp 71-5’NeoR-CreERT2-IRES-PuroR-FRT)在冰浴混合10min。
C.3 x 10 5ES细胞和20ul RNP与DNA模板混合，在电转杯(BioRad,cata:1652081)中进行电转染。电转染条件：1200伏，脉冲间隔30毫秒。电转染后，在存在滋养细胞(sigmaaldrich Cata:PMEF-CFX)的10厘米培养皿中培养用培养液稀释的ES细胞。ES细胞的培养基和培养条件参照ATCC关于该细胞系的说明。
D.持续培养细胞ES48小时后，在培养基加入1ug/ml Puromycin(Thermofisher Cata:A1113802)。连续培养7天。
E.挑取单克隆ES细胞，并转移到48孔培养板中继续生长。3天后，用移液器枪头打散ES细胞，其中一半细胞用于提取基因组DNA，另外一半细胞冻存在48孔细胞培养板备用。
F.分别在插入片段的5’端和3’端和插入位点的衔接处设计引物，利用PCR方法筛选定点整合了表达盒的基因组DNA。引物序列如下:Insert 5F:GCAGTCTGCACTCTCTGTGG；Insert 5R:tcgagggacctaTACCGTTC；Insert 3F:cgtgacatgtgagcaaaagg；Insert 3R：GGAACCTTTCCCCCATAAAA。引物设计原理和筛选结果如说明书附图中的图8。经PCR鉴定，28个克隆中有15个克隆是PCR阴性，有9个克隆插入片段的5‘端和3'端都为阳性，还有少数克隆只是在一个末端为PCR阳性。结果显示，利用CRISPR/Cas9技术，外源DNA定点插入受体细胞的比例达到30％。将经基因修饰的ES克隆命名为ES 5’Neo-Loxp 71。
利用同样的策略，本申请制备了另外一个ES克隆，被插入的基因结构是WT-Loxp-hprt intron-5'Neo-PGKpromoter-rBGpA-EF1a-CreERT2-IRES-puro-FRT。将此经修饰后的ES克隆命名ES 5’Neo-WTLoxp。
3.酵母原生质球介导的细胞融合技术
大片段DNA定向插入受体细胞的难点在于使用传统的方法，如脂质体转染，电转等，转化效率低，线形大片段形式的外源DNA容易断裂，同时从酵母细胞纯化大片段DNA的效率非常低。对此，本申请选用了酵母原生质球融合技术。在PEG的介导下,酵母原生质球可以和细胞融合，从而将大片段DNA导入受体细胞。虽然现有技术中报道了酵母原生质球介导的细胞融合可以用于介导将含有大片段DNA的YAC导入受体细胞，但是这种导入多属于染色体随机插入，而不是定点插入。同时，线形的YAC载体容易在细胞内降解或断裂。对此，本申请进行了改进，本申请将YAC载体拼接成环状的YAC载体，同时，将大片段DNA装载到环状的YAC载体中，从而提高了完整基因定点插入细胞染色体的效率。另外，为了保持原生质融合后ES细胞的多功能性，我们还选用了以129S5/Sv为遗传背景的胚胎干细胞(ES细胞)。这种遗传背景的ES细胞比其他遗传背景的ES细胞更不容易在体外培养时产生分化。
具体方法步骤:
A.制备酵母原生质细胞:
将装载大片段IGL(携带TAR-IGL-Loxp 66基因)的酵母菌液加入到含有0.2mg/mL Hygromycin的YPD(Sigma,Cata:Y1375)培养基中，30℃摇菌过夜。第二天，用YPD培养基10倍稀释菌液，继续摇菌3-4小时，直到OD600值2.0-3.0。随后以2500g离心15min，收集酵母菌。用20ml1M山梨醇悬浮酵母细胞沉淀，在4℃静置2小时。离心收集酵母细胞，用SPEM缓冲液(1M山梨醇,0.1M磷酸钠缓冲液混合物pH7.4,和10mM EDTA,pH 8.0)混匀细胞沉淀,再加75ul 14mM 2-ME和100UL Zymolase进行去壁处理，30℃消化30分钟。用1M山梨醇洗涤消化后的细胞。随后将细胞悬浮于2ml的STC溶液(1M sorbitol,10mM Tris-HCl,pH 7.5,10mMCaCl 2和2.5mM MgCl 2)备用。
B.PEG介导的细胞融合:
与此同时收集上述经过基因修饰的ES细胞(例如ES 5'Neo-Loxp 71)。将ES 5'Neo-Loxp 71细胞与TAR-IGL-Loxp 66酵母细胞以1：100混合于PBS溶液。室温离心，细胞混合液悬浮于含有10％DMSO的PEG1500溶液(Roche Applied Science,cata:10783641001)。两分钟后，用培养液悬浮融合后的ES细胞，200g离心5分钟。悬浮细胞混合液，用ES细胞培养基稀释2x10 6ES细胞，在5个10cm培养皿培养。在50％ES细胞贴壁之后，加入Tamoxifen(1μg/ml)(Sigma，Cata：T5648-1G)诱导ES细胞表达Cre重组酶。16小时后，洗去Tamoxifen，加入G418，筛选抗性克隆。
作为对照，用PEG的方法将ES 5'Neo-WTLoxp细胞与TAR-IGL-WTLoxp酵母细胞加以融合，随后用G418筛选，但是未观察到G418抗性克隆生成，说明WT Loxp不能介导大片段环状DNA的定点插入，或者插入效率很低。
因此，本申请也尝试了常规的Cre-Lox重组方法，即使用野生型的Loxp序列，但没能将大分子DNA稳定地定点整合到细胞染色体。这可能是因为Cre催化的Loxp序列依赖的重组反应是可逆的，即Loxp位点之间可能发生反向重组或删除，因此野生型Loxp位点之间的重组不能有效地介导大分子DNA稳定地定点整合到细胞染色体。
C.阳性基因插入细胞的检测:
在G418的存在下连续培养一个星期之后，挑取表达GFP的G418抗性单克隆ES细胞(ES 5'Neo-WTLoxp)，转移到48孔培养板继续生长。3天后，用移液器枪头打散ES细胞，其中的一半细胞用于提取基因组DNA，而将另外一半细胞冻存在48孔细胞培养板备用。考虑到具有G418抗性的克隆会在外源片段的Loxp 66位点和染色体Loxp 71位置之间发生重组，从而在插入片段的两端生成WTLoxp位点和Loxp 71/66位点，因此在插入位点WTLoxp和Loxp 71/66的两端设计引物，用PCR方法鉴定定点插入ES克隆。
引物序列如下:
WT Loxp F:GGAGATCCTCAGGTCATTGC；
WT Loxp R:GGCAGAGCTTTGCTTTTGTT；
Loxp 66/71 F:CCTTGACCCAGAAATTCCAC；
Loxp 66/71 R:TGGAGGCCATAAACAAGAAGA。引物设计及其PCR鉴定结果如说明书附图中的图9。
经过G418筛选，所有细胞克隆都是Loxp 71/66 PCR结果阳性，其中75％的细胞克隆是Loxp 71/66和WT Loxp双阳性，而25％的细胞克隆只有Loxp71/66 PCR单阳性而WT Loxp PCR是阴性的，说明在WT Loxp位点可能发生了第二次基因重组导致了多拷贝插入或者发生了基因断裂。可以用PCR的方法对这种情况予以排除，从而进一步挑选双阳性克隆并进行插入基因IGL完整性的鉴定。所用的PCR引物见说明书附图中的图6。鉴定结果显示所有的PCR结果是阳性，表明插入基因完整。
D.删除选择性标记:
随着外源基因IGL插入染色体，细胞会表达一些不必要的选择性基因，如HygR,NeoR，CreERT2和PuroR等。这些选择性基因可以利用FLP/FRT重组加以删除。如说明书附图中的图3显示。合成FLP质粒(pCAG-Flpe)序列(Addgene,Cata:13787)。利用常规脂质体转化方法，将pCAG-Flpe质粒转入插入了IGL基因的ES克隆，挑选GFP阴性，G418敏感和Puromycin敏感的ES克隆备用。
Cre-Lox重组是一种用于在细胞DNA的特定位点上进行删除、插入、转座和倒位操作的位点特异性重组酶技术。loxP位点由34bp的特殊位点序列组成，中间8bpDNA碱基是不对称序列，决定了loxP序列的方向，不对称序列两边是两段13bp反向对称序列，决定了与Cre的结合效率。当对称序列的一端发生突变，如Loxp 71(5’端对称序列存在突变)和Loxp 66(3’端对称序列存在突变)，Cre仍然可以催化突变Loxp之间(如Loxp 71和Loxp 66)发生重组反应。不过在Loxp 71和Loxp 66发生重组反应后，产生野生型Loxp和双突变Loxp(Loxp 71/66)。因为Loxp 71/66在两端的对称序列都存在突变，其和Cre的结合能力大为降低，所以避免了WT Loxp和Loxp 71/66之间发生反向重组反应，从而增强了插入基因的稳定性。本申请通过以上实验进行比较发现这种突变型Loxp，而非野生型Loxp，有助于大片段DNA的定点插入。
本申请通过实验发现，对于介导大分子DNA稳定地定点整合到细胞染色体，Loxp双突变Loxp66/71是最优选的方案。相对于野生型Loxp，或者含1个突变的Loxp，例如Loxp/Loxp66或者Loxp/Loxp71，或者含其他突变位置的Loxp双突变，loxp66/71的效果最好，转化效率最高，能最有效最稳定地将大分子DNA稳定地定点整合到细胞染色体，最适合于大片段DNA的定点整合和克隆。
本申请涉及的序列如下：
1.序列1 SEQ ID NO:1
loxp66序列：
Figure PCTCN2020118294-appb-000002
2.序列2 SEQ ID NO:2
WT loxp序列
Figure PCTCN2020118294-appb-000003
3.序列3 SEQ ID NO:3
用于载体TAR-loxp66末端和IGL1连接处PCR产物1的正向引物
Figure PCTCN2020118294-appb-000004
4.序列4 SEQ ID NO:4
用于载体TAR-loxp66末端和IGL1连接处PCR产物1的反向引物
Figure PCTCN2020118294-appb-000005
5.序列5 SEQ ID NO:5
用于IGL1内部PCR产物2的正向引物
Figure PCTCN2020118294-appb-000006
6.序列6 SEQ ID NO:6
用于IGL1内部PCR产物2的反向引物
Figure PCTCN2020118294-appb-000007
7.序列7 SEQ ID NO:7
用于IGL1和IGL2的连接处PCR产物3的正向引物
Figure PCTCN2020118294-appb-000008
8.序列8 SEQ ID NO:8
用于IGL1和IGL2的连接处PCR产物3的反向引物
Figure PCTCN2020118294-appb-000009
9.序列9 SEQ ID NO:9
用于IGL2内部PCR产物4的正向引物
Figure PCTCN2020118294-appb-000010
10.序列10 SEQ ID NO:10
用于IGL2内部PCR产物4的反向引物
Figure PCTCN2020118294-appb-000011
11.序列11 SEQ ID NO:11
用于IGL2和IGL3连接处PCR产物5的正向引物
Figure PCTCN2020118294-appb-000012
12.序列12 SEQ ID NO:12
用于IGL2和IGL3连接处PCR产物5的反向引物
Figure PCTCN2020118294-appb-000013
13.序列13 SEQ ID NO:13
用于IGL3内部PCR产物6的正向引物
Figure PCTCN2020118294-appb-000014
14.序列14 SEQ ID NO:14
用于IGL3内部PCR产物6的反向引物
Figure PCTCN2020118294-appb-000015
15.序列15 SEQ ID NO:15
用于IGL3和载体TAR-loxp66末端连接处PCR产物7的正向引物
Figure PCTCN2020118294-appb-000016
16.序列16 SEQ ID NO:16
用于IGL3和载体TAR-loxp66末端连接处PCR产物7的反向引物
Figure PCTCN2020118294-appb-000017
17.序列17 SEQ ID NO:17
用于筛选整合了表达盒的基因组DNA的PCR的引物Insert 5F:
Figure PCTCN2020118294-appb-000018
18.序列18 SEQ ID NO:18
用于筛选整合了表达盒的基因组DNA的PCR的引物Insert 5R:
Figure PCTCN2020118294-appb-000019
19.序列19 SEQ ID NO:19
用于筛选整合了表达盒的基因组DNA的PCR的引物Insert 3F:cgtgacatgtgagcaaaagg
20.序列20 SEQ ID NO:20
用于筛选整合了表达盒的基因组DNA的PCR的引物Insert 3R
Figure PCTCN2020118294-appb-000020
21.序列21 SEQ ID NO:21
用于鉴定WT loxp阳性的引物WT loxp F:
Figure PCTCN2020118294-appb-000021
22.序列22 SEQ ID NO:22
用于鉴定WT loxp阳性的引物WT loxp R:
Figure PCTCN2020118294-appb-000022
23.序列23 SEQ ID NO:23
用于鉴定Loxp66/71阳性的引物Loxp66/71F:
Figure PCTCN2020118294-appb-000023
24.序列24 SEQ ID NO:24
用于鉴定Loxp66/71阳性的引物Loxp66/71R:
Figure PCTCN2020118294-appb-000024
25.序列25 SEQ ID NO:25
TAR-loxp66序列
Figure PCTCN2020118294-appb-000025
Figure PCTCN2020118294-appb-000026
Figure PCTCN2020118294-appb-000027
Figure PCTCN2020118294-appb-000028
Figure PCTCN2020118294-appb-000029
Figure PCTCN2020118294-appb-000030
26.序列26 SEQ ID NO:26
loxp71-hprt intron-5'Neo-PGK promoter-rBGpA-EF1a-CreERT2-IRES-puro-FRT序列
Figure PCTCN2020118294-appb-000031
Figure PCTCN2020118294-appb-000032
Figure PCTCN2020118294-appb-000033
Figure PCTCN2020118294-appb-000034
Figure PCTCN2020118294-appb-000035
Figure PCTCN2020118294-appb-000036
Figure PCTCN2020118294-appb-000037

Claims (10)

  1. 整合目的基因片段的方法，其包括以下步骤：
    1)获得目的基因片段；
    2)将目的基因片段克隆到载体中，在所获得的包含目的基因片段的载体中插入了抗性表达盒1，优选截断型新霉素(G418)抗性表达盒1，所述表达盒1在5’至3’方向上包含突变型Loxp 1，优选Loxp66序列、部分HPRT内含子和抗性基因a部分，优选新霉素抗性基因Neo基因3’端部分；
    3)将含有所述目的基因片段的载体转化到微生物细胞内，优选为酵母，尤其是酿酒酵母(saccharomyces cerevisiae)细胞内；
    4)将所述微生物内含有目的基因片段的所述载体导入到受体细胞，其中所述受体细胞的基因组中已经定点导入了抗性表达盒2，优选截断型新霉素(G418)抗性表达盒2，所述表达盒2在5’至3’方向上包含部分HPRT内含子、突变型Loxp 2，优选Loxp71序列、抗性基因b部分，优选Neo基因5’端部分、含有雌激素受体(estrogen receptor，ER)的配体结合区突变体(ERT2)与Cre重组酶的融合蛋白CreERT2，和IRES-PuroR结构；
    5)诱导CreERT2的重组酶活性，优选通过加入Tamoxifen诱导CreERT2的重组酶活性，所述Cre重组酶介导所述目的基因片段定点整合到所述受体细胞的基因组中，同时所述表达盒1和表达盒2形成表达完整抗性基因，优选新霉素的抗性表达盒；
    6)筛选基因组中定点整合了所述目的基因片段的所述受体细胞。
  2. 根据权利要求1的方法，其中在所述步骤2)中的抗性表达盒1中所包含的抗性基因a部分，优选新霉素抗性基因Neo的3’端部分和所述步骤4)中的抗性表达盒2中的抗性基因b部分，优选Neo基因5’端部分是功能互补的，优选地，所述Neo基因5’端部分和所述Neo的3’端部分是在92位截断的，更优选地，新霉素的第92位氨基酸是由所述Neo基因5’端部分的末尾核苷酸和Neo基因的3’端部分的起始核苷酸共同编码的，更优选地，新霉素的第92位氨基酸是由所述Neo基因5’端部分的末尾2个核苷酸和Neo基因的3’端部分的起始处第一个核苷酸共同编码的，
    并且所述步骤6)通过加入特异性针对所述抗性基因的试剂，优选抗生素 G418来筛选基因组中定点整合了所述目的基因片段的所述受体细胞。
  3. 根据权利要求1或2的方法，其中在所述步骤1)中的所获得的包含目的基因片段的载体中，所述目的基因片段的上游存在FRT序列，同时在所述步骤4)中的表达盒中，在所述IRES-PuroR结构的下游存在FRT序列，
    并且在所述步骤6)之后还包括步骤7)：将包含FLP的质粒转入所述受体细胞以除去所述表达盒。
  4. 根据前述权利要求中任一项的方法，其中所述步骤1)中的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。
  5. 根据前述权利要求中任一项的方法，其中所述步骤2)中所获得的包含目的基因片段的载体是环状载体。
  6. 根据前述权利要求中任一项的方法，其中所述步骤2)中的所述载体是线性化载体，所述线性化载体优选为拥有被克隆目的DNA片段同源末端序列的线性化载体，更优选为线性化酵母穿梭载体。
  7. 根据权利要求5的方法，其中所述环状载体是通过同源重组拼接技术(优选酵母的转化偶联重组(TAR)技术)，或者同源重组拼接技术(优选酵母的转化偶联重组(TAR)技术)与基因组编辑技术的组合获得的。
  8. 根据前述权利要求中任一项的方法，其中在所述步骤4)中使用酵母原生质球融合(yeast protoplast fusion)技术将将所述微生物内含有目的基因片段的所述载体导入到受体细胞。
  9. 根据前述权利要求中任一项的方法，其中所述步骤2)中的抗性表达盒1，优选截断型新霉素(G418)抗性表达盒1中的所述突变型Loxp 1和/或所述步骤4)中的抗性表达盒2，优选截断型新霉素(G418)抗性表达盒2中所述突变型Loxp 2用于将所述表达盒及其下游大片段DNA定点稳定地导入所述受体细胞的基因组中。
  10. 根据前述权利要求中任一项的方法，其中所述步骤2)中的抗性表达盒1，优选截断型新霉素(G418)抗性表达盒1中包含的HPRT内含子、抗性基因a部分，优选Neo基因3’端部分和所述步骤4)中的抗性表达盒2，优选截断型新霉素(G418)抗性表达盒2中包含的HPRT内含子、抗性基因b部分，优选Neo基因5’端部分，以及步骤5)中形成的表达完整抗性基因的抗性表达盒， 优选新霉素Neo的新霉素(G418)抗性表达盒，用于筛选目的基因片段定点整合后的细胞重组子。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023073738A1 (en) * 2021-10-29 2023-05-04 Universita' Degli Studi Di Trento Genetic construct for tracking and/or ablating quiescent cells
CN118460446A (zh) * 2024-07-12 2024-08-09 内蒙古自治区农牧业科学院 一种支原体突变株及其制备方法与应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006333742A (ja) * 2005-05-31 2006-12-14 Chemicals Evaluation & Research Institute 細胞及び細胞へのｄｎａ導入方法
CN101684476A (zh) * 2008-09-22 2010-03-31 中国科学院上海生命科学研究院 转基因构建物及其在制备时空可调性肝脏损伤模型中的应用
CN103361342A (zh) * 2012-03-30 2013-10-23 上海杰隆生物工程股份有限公司 一种转基因定位整合的方法及其应用
CN107177616A (zh) * 2017-05-26 2017-09-19 华南农业大学 一种多基因组装载体系统及其多基因组装方法
WO2018129203A2 (en) * 2017-01-06 2018-07-12 The Regents Of The University Of California Method for temporal and tissue-specific drug delivery and induced nucleic acid recombination

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001086989A (ja) * 1999-09-17 2001-04-03 Univ Osaka 哺乳類型Ｃｒｅリコンビナーゼ遺伝子
CN103215295B (zh) * 2013-04-11 2015-04-22 西北农林科技大学 一种β-酪蛋白位点定点整合Lys基因的打靶载体及其构建的细胞
CN106978416B (zh) * 2016-01-18 2020-11-06 上海转基因研究中心 一种基因定位整合表达系统及其应用
CN107119067B (zh) * 2017-04-25 2019-10-18 华南理工大学 谷氨酸棒杆菌中一种基因连续无痕敲除的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006333742A (ja) * 2005-05-31 2006-12-14 Chemicals Evaluation & Research Institute 細胞及び細胞へのｄｎａ導入方法
CN101684476A (zh) * 2008-09-22 2010-03-31 中国科学院上海生命科学研究院 转基因构建物及其在制备时空可调性肝脏损伤模型中的应用
CN103361342A (zh) * 2012-03-30 2013-10-23 上海杰隆生物工程股份有限公司 一种转基因定位整合的方法及其应用
WO2018129203A2 (en) * 2017-01-06 2018-07-12 The Regents Of The University Of California Method for temporal and tissue-specific drug delivery and induced nucleic acid recombination
CN107177616A (zh) * 2017-05-26 2017-09-19 华南农业大学 一种多基因组装载体系统及其多基因组装方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NISHIHAMA RYUICHI, ISHIDA SAKIKO, URAWA HIROKO, KAMEI YASUHIRO, KOHCHI TAKAYUKI: "Conditional Gene Expression/Deletion Systems for Marchantia polymorpha Using its Own Heat-Shock Promoter and Cre/ lox P-Mediated Site-Specific Recombination", PLANT AND CELL PHSIOLOGY, OXFORD UNIVERSITY PRESS, UK, vol. 57, no. 2, 1 February 2016 (2016-02-01), UK, pages 271 - 280, XP055798761, ISSN: 0032-0781, DOI: 10.1093/pcp/pcv102 *
QIU SHUPING, CHEN ZAI-JIE, WANG FENG: "Application of Cre/loxp Site-Specific Recombination Technology on Transgenic Plants", FUJIAN NONGYE XUEBAO -FUJIAN JOURNAL OF AGRICULTURAL SCIENCES, FUJIAN SHENG NONGKEYUAN, CN, vol. 23, no. 2, 1 January 2008 (2008-01-01), CN, pages 211 - 217, XP055798760, ISSN: 1008-0384, DOI: 10.19303/j.issn.1008-0384.2008.02.020 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023073738A1 (en) * 2021-10-29 2023-05-04 Universita' Degli Studi Di Trento Genetic construct for tracking and/or ablating quiescent cells
CN118460446A (zh) * 2024-07-12 2024-08-09 内蒙古自治区农牧业科学院 一种支原体突变株及其制备方法与应用

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