WO2015158115A1 - 一种简便高效的基因编辑方法 - Google Patents

一种简便高效的基因编辑方法 Download PDF

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WO2015158115A1
WO2015158115A1 PCT/CN2014/087814 CN2014087814W WO2015158115A1 WO 2015158115 A1 WO2015158115 A1 WO 2015158115A1 CN 2014087814 W CN2014087814 W CN 2014087814W WO 2015158115 A1 WO2015158115 A1 WO 2015158115A1
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gene
nucleic acid
seq
tesc
fragment
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马延和
刘谊兰
邢建民
王钦宏
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中国科学院天津工业生物技术研究所
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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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers

Definitions

  • the invention belongs to the field of genetic engineering, and in particular relates to a gene editing method.
  • a cleavage site upstream of the two-way screening marker gene URA3 of yeast improves the efficiency of seamless editing
  • Nucleic Acids research, 2010.38(8): p.2570-2576 developed a variety of bidirectional screening marker genes galK, thyA, tolC, tetA-sacB in E. coli (Warming et al., Simple and highly efficient BAC recombineering using galK selection.
  • the difficulty of gene editing technology so far, that is, the efficient deletion of marker genes in the two-way screening system, has not been effectively solved.
  • the deletion of the traditional two-way screening marker gene firstly transforms the single-stranded or double-stranded nucleic acid fragment containing the homologous region upstream and downstream of the insertion site into the cell, and then replaces the resistance marker gene on the genome by homologous recombination, and finally passes through the culture.
  • the negative sieve substrate was added to the base to complete the enrichment of the recombinant cells, and the cells for removing the screening marker were selected to obtain the cells without trace editing. Therefore, the traditional gene editing technology requires two transformations.
  • the first transformation is used to transfer the nucleic acid fragment carrying the gene of interest and the marker gene into the cell for homologous recombination, and the target gene and the marker gene enter the target genome after homologous recombination;
  • the second transformation is used to delete the marker gene, ie, the nucleic acid fragment containing the homologous region upstream and downstream of the insertion site is transferred into the cell, and the resistance marker gene on the genome is replaced by homologous recombination.
  • the efficiency of deleting marker genes in this way is often very low.
  • the two-way screening system for screening recombinants in gene editing technology comprises positive screening and negative screening.
  • the positive screening generally confers antibiotic resistance to the antibiotic by the antibiotic resistance marker gene or the corresponding selective defect culture by the auxotrophic gene. The nature of basal growth is achieved.
  • Negative screening generally utilizes the sensitivity of certain genes to recombinants for certain substances, such as positive/negative screening using the sacB-neo fusion gene, neo (kanamycin) resistance. Used for positive screening, and Sucrose toxicity due to expression of the sacB gene was used for negative selection (Warming et al., Simple and highly efficient BAC recombineering using galK selection. Nucleic acids research, 2005. 33(4): p.e36).
  • Two-way screening can also be achieved by a two-way screening of marker genes.
  • the tolC gene can be used for both positive and negative screening (Gregg et al., Rational optimization of tolC as a powerful dual selectable marker for genome engineering. Nucleic acids research, 2014.DOI: 10.1093/nar/gkt1374).
  • the success rate of negative screens is often extremely low or often fails. This is mainly because the lethal process of negative screening requires the production and accumulation of toxic substances. During the accumulation process, the cells are prone to spontaneous mutations and produce resistance to toxic substances, so that the cells can survive while retaining negative screening genes.
  • the conversion process before negative screening often introduces exogenous cells that are insensitive to negative screening materials, leading to failure of the negative screening process.
  • the present invention provides a gene editing method comprising the following steps:
  • the parallel repeat sequence on the nucleic acid fragment in step b above is the original sequence upstream or downstream of the target nucleic acid to be modified.
  • the parallel repeats on the nucleic acid fragments in step b are homologous to sequences upstream or downstream of the target nucleic acid immediately adjacent to the modification site to achieve seamless editing.
  • the recombinant cell selected in step c is cultured in a medium containing no positive or negative screening material in step d to allow parallel repeats on the nucleic acid fragment and upstream or downstream of the site to be modified. Homologous recombination occurs in the corresponding parallel repeats, thereby deleting the marker gene.
  • the bidirectional screening marker gene in step b comprises one or more Chi sites to increase the efficiency of bidirectional screening marker gene deletion.
  • the Chi site can be introduced into any one or more of the two-way screening marker genes without affecting the function of the two-way screening marker gene.
  • the Chi site may be forward or reverse, and multiple Chi sites may be continuous or discontinuous. Different Chi sites can be used in different types of genomes, and different intensity Chi site sequences can be used in the same type of genome.
  • the method of the invention is used for the editing of genes at any position in the prokaryotic or eukaryotic genome, ie the target nucleic acid is the genome of a prokaryotic or eukaryotic cell.
  • the methods of the invention are used for gene editing on a plasmid or artificial chromosome (BAC), i.e., the target nucleic acid is a plasmid or an artificial chromosome.
  • BAC plasmid or artificial chromosome
  • Also provided in a specific embodiment of the invention is an artificial sequence for homologous recombination, the sequence being SEQ ID NO.: 1, SEQ ID NO.: 22, SEQ ID NO.: 24, SEQ ID NO .:27 or SEQ ID NO.:28.
  • a method for homologous recombination comprising the steps of: a. providing a target cell E. coli MG1655, the target cell comprising a target nucleic acid fadE gene carrying a site to be modified; b. Using the fadE I gene comprising the homologous region at both ends of fadE, the 5'-end parallel repeat and cat-sacB, the sequence of which is SEQ ID NO.:1, transforming E.coli MG1655 competent target cells containing pKD46, Homologous recombination of the nucleic acid fadEI gene and the target nucleic acid fadE; c.
  • MG1655 ⁇ fadE positive screening by LB solid medium coated with chloramphenicol after incubation to obtain recombinant MG1655 ⁇ fadE::cat-sacB; d. Purified MG1655 ⁇ fadE ::cat-sacB was negatively screened by LB liquid medium containing 10% sucrose; e. PCR detection by PCR primers, and sequencing confirmed to obtain a trace-free deletion strain.
  • a further embodiment of the present invention provides a method for homologous recombination, comprising the steps of: a. providing a target cell E. coli MG1655, the target cell comprising a target nucleic acid tesC gene to be deleted; b. using PCR
  • the esC I fragment was obtained by using the E. coli MG1655 genome as a template, using the upstream and downstream primers, the tesCp1 sequence of SEQ ID NO.: 4 and the tesCp2 of SEQ ID NO.: 5 to amplify the E. coli tesC gene and the upstream and downstream partial sequences. This fragment was obtained with pMD18-T pMD18-tesC; c.
  • coli DH5 ⁇ strain obtaining pMD-tesC-cat-chi-sacB plasmid; f. using PCR reaction to pMD-tesC- cat-chi-sacB is a template, using upstream and downstream primers, the sequence is SEQ ID NO.: 4 tesCp1 and the tesCp2 sequence of SEQ ID NO.: 5 were amplified to obtain the tesC IV fragment; g. fragment tesC IV electrotransformed E. coli MG1655 competent cells containing pKD46, and incubated with LB solids containing chloramphenicol after incubation The medium was positively screened to obtain recombinant MG tesC::cat-chi-sacB; h.
  • MG tesC :cat-chi-sacB and negatively screened by LB liquid medium containing 10% sucrose; i.
  • the tesCp1 of SEQ ID NO.: 4 and the tesCp2 of SEQ ID NO.: 5 were primer PCR assays, and sequencing confirmed that a deletion-free strain was obtained.
  • Figure 1 Three main ways of gene editing.
  • Figure 2 Schematic diagram of the principle of parallel repeat sequence assisted gene editing.
  • Figure 3 Schematic representation of a two-way screening marker gene containing a Chi site.
  • Figure 4 is a schematic diagram showing the principle of parallel repeat and Chi site assisted gene editing.
  • Figure 5 Schematic representation of gene editing in E. coli using the method of the invention.
  • Figure 6 Plasmid pMD18-T map (purchased from TaKaRa).
  • Figure 7 Plasmid pEASY-cat-sacB map.
  • Figure 8 Map of plasmid pKD46.
  • Figure 9 Sequence of FADEI showing the FADE upstream homologous region, cat-sacB, FADE upstream homologous region, and FADE downstream homologous region, respectively.
  • Figure 10 Sequence of FAA-URA3 showing the homologous region upstream of the faa1 start codon, the homologous region upstream of the faa1 start codon, and the homologous region downstream of the faa1 stop codon.
  • Figure 11 Sequence of pMD-ldhAI-cat-sacB-FAR showing the sequence upstream of the insertion site, cat-sacB, homologous region upstream of the insertion site, FAR, homologous region downstream of the insertion site.
  • Figure 12 Sequence of pMD-tesC-cat-sacB-FAR showing homologous regions upstream of tesC, cat-sacB, homologous regions upstream of tesC, homologous regions downstream of FAR and tesC, respectively.
  • the invention utilizes the principle of restarting the replication fork by homologous recombination after high-frequency spontaneous rupture of the genome, and realizes the process of gene editing by one-step transformation and two-step recombination by means of serially repeating sequences in parallel at both ends of the marker gene.
  • the traditional gene editing method requires two transformations
  • the method provided by the present invention can realize the expected transformation and the deletion of the two-way screening marker gene by only one transformation, the operation of gene editing is greatly simplified. step.
  • the method provided by the present invention improves the deletion efficiency of the two-way screening marker gene.
  • the efficiency of deletion of the two-way screening marker gene may be due to the traditional deletion of the two-way screening marker gene by homologous recombination between molecules, which is largely affected by transformation efficiency (generally not More than 1/1000), and the method of the invention is accomplished by intramolecular recombination, each cell containing a fragment that undergoes homologous recombination.
  • the method of the present invention increases the efficiency of negative screening by introducing a Chi site in a bidirectionally screened marker gene, up to 50%. Since the method of the present invention can perform seamless editing of genes, operations such as deletion, substitution, and insertion of genes can be continuously performed.
  • target cell refers to a cell to be transformed, which contains a target nucleic acid, which is transformed with a nucleic acid fragment designed and constructed according to the present invention to cause homologous recombination of the nucleic acid fragment with the target nucleic acid, Thereby achieving the required gene editing.
  • target nucleic acid refers to a nucleic acid that requires gene editing, and the target nucleic acid may be a genome of a prokaryotic cell or a eukaryotic cell, or may be a plasmid or an artificial chromosome (BAC) or the like.
  • BAC artificial chromosome
  • nucleic acid includes DNA, cDNA, and RNA.
  • gene editing refers to the alteration of a gene sequence at a site to be modified, including gene deletion, insertion and substitution.
  • Gene editing in the context of the present invention involves editing a gene or any base, i.e., deleting, inserting or replacing the original gene or base on the target nucleic acid with a gene or any base.
  • non-marking editing or “no trace gene editing” refers to the absence of unwanted extra bases on the edited nucleic acid.
  • site to be modified or “modification site” refers to a specific location on a target nucleic acid that requires genetic editing.
  • homologous refers to the nucleotide sequence similarity or identity between two nucleic acids.
  • homology can be assessed by DNA-DNA or DNA-RNA hybridization (eg, Nucleic Acid Hybridization by Haines and Higgins (editor) (IRL Press, Oxford, UK). Said), or by comparing the sequence identity between two nucleic acids for the assessment of homology.
  • homologous means having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98 between two nucleic acids.
  • Sequence similarity or consistency of % 99% or 100%, preferably having sequence similarity of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% Or consistency, more preferably having 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistency, most preferably 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistency.
  • Software that can be used for sequence alignments such as the BLAST and ClustalW programs, both of which can be found online.
  • nucleic acid fragment constructed according to the invention refers to an upstream and downstream homologous sequence comprising a site to be modified, a gene of interest (when used for gene deletion, no gene of interest) , a parallel repeat and a two-way screening of a nucleic acid fragment of the marker gene.
  • a sequence homologous to the upstream and downstream sequences of the site to be modified is located at both ends of the nucleic acid fragment for homologous recombination with the target nucleic acid.
  • gene of interest refers to a gene used for gene editing at a site to be modified, for example, by replacing the original gene on the target nucleic acid at the site to be modified or inserted into the target nucleic acid, when used to delete the gene on the target nucleic acid or In the case of a base, the gene of interest is not required, that is, the gene of interest is absent on the nucleic acid fragment constructed by the present invention.
  • parallel repeat refers to a sequence on a nucleic acid fragment constructed in accordance with the present invention that is homologous to an upstream or downstream sequence of a site to be modified on a target nucleic acid, for example, the 5' end of the nucleic acid fragment constructed in accordance with the present invention contains a position to be modified.
  • a downstream parallel (ie, 3' end) homologous parallel repeat ie, a corresponding parallel repeat downstream of the site to be modified
  • the 3' end of the nucleic acid fragment constructed in accordance with the invention contains upstream of the site to be modified (ie, 5' end) homologous parallel repeats (ie, corresponding parallel repeats upstream of the site to be modified).
  • parallel repeats This sequence on the nucleic acid fragment constructed by the present invention and the sequence homologous thereto on the target nucleic acid are referred to as "parallel repeats" in the present invention.
  • the length of the parallel repeat sequences can be variable, for example, in the range of 30 bases to 1000 bases, and one skilled in the art can determine the length of the parallel repeat sequences according to specific needs.
  • the term "two-way screening marker gene” or “marker gene” refers to a gene that can perform both positive and negative screening, and can be one or more genes, for example, tolC, galK, thyA, neo-sacB, tetA-sacB. , cat-sacB, etc.
  • “Positive screening” has the meaning commonly understood by those skilled in the art, that is, generally achieved by the antibiotic resistance marker gene conferring resistance to the antibiotic to the recombinant or by the auxotrophic gene conferring characteristics in the corresponding selective defective medium, For example, a positive screen kanamycin or chloramphenicol, a uracil-deficient medium or the like is used.
  • Negative screening has the meaning commonly understood by those skilled in the art, that is, it is generally characterized by the presence of a negative screen having adverse effects on cells under specific defined conditions, such as cell death or inhibition/suppression of cell growth.
  • the most commonly used negative selection marker is sacB of Bacillus subtilis. When sacB is introduced into a heterologous host such as E. coli, it can cause cell death in the presence of exogenous sucrose.
  • a “Chi locus” or “Chi sequence” is a short DNA fragment in the genome in which the probability of homologous recombination increases.
  • the sequence of the Chi locus in the genome of different types of organisms may be different.
  • Figure 1 shows the three main ways of gene editing, namely the deletion, substitution or insertion of a gene or base.
  • Figure 2 is a schematic diagram of the principle of parallel repeat sequence assisted gene editing.
  • a nucleic acid fragment designed and constructed according to the present invention contains a sequence homologous to both upstream and downstream of a site to be modified at both ends thereof, and thus the fragment is capable of interacting with a target nucleic acid (genome) after being transformed into a cell. Or homologous recombination occurs, the nucleic acid fragment is integrated into the target nucleic acid, the gene or base of the site to be modified is deleted, the gene or base of the site to be modified is replaced, or inserted into the site to be modified.
  • the recombinant is enriched by positive screening, for example, if one of the two-way screening marker genes on the nucleic acid fragment is cat, that is, a chloramphenicol resistance gene, chlorine may be used.
  • the medium of themycin screens the transformed cells to enrich for recombinant sub-cells that have undergone homologous recombination to have chloramphenicol resistance.
  • the method of the invention utilizes homologous recombination between two parallel repeats Marker genes are eliminated.
  • the parallel repeats are located one upstream or downstream of the site to be screened, and on a nucleic acid fragment constructed according to the invention, a two-way screening marker gene is present between the two parallel repeats.
  • nucleic acid fragment constructed according to the present invention contains a parallel repeat at the 5' end
  • sequence is homologous to the 3' upstream sequence of the site to be modified
  • nucleic acid fragment constructed by the present invention contains a parallel repeat at the 3' end
  • This sequence is homologous to the 5' upstream sequence of the site to be modified, such that when the two parallel repeats are located within the same molecule, homologous recombination can occur and the marker gene between them is deleted.
  • the parallel repeat on the nucleic acid fragment is the original sequence upstream or downstream of the target nucleic acid to be modified.
  • the length of the parallel repeats can be varied as desired, for example, in the range of 30 to 1000 bases, and one skilled in the art can determine the length of the parallel repeats as needed.
  • the target nucleic acid contains the two parallel repeats, and the two parallel repeats are separated by a marker gene, and then the two parallel repeats are allowed to undergo intramolecular homology.
  • the source is recombined, for example, by culturing the recombinant daughter cells in a medium containing no positive or negative screening material for a period of time to remove the marker gene.
  • the method of the invention allows for the seamless editing of genes. Still referring to Figure 2, if the parallel repeats on the nucleic acid fragments constructed in accordance with the present invention are homologous to sequences upstream or downstream of the target nucleic acid immediately adjacent to the modification site, seamless editing can be achieved. "Sequence immediately upstream or downstream of a modification site” means that the upstream or downstream sequence is contiguous with the modification site, with no base spacing.
  • the introduction of one or more Chi sites in the bidirectionally screened marker gene further increases the efficiency of bidirectional screening marker gene deletion (see Figures 3 and 4), resulting in a final negative screening efficiency of up to 50%.
  • the Chi site can be introduced into any one or more of the two-way screening marker genes without affecting the function of the two-way screening marker gene.
  • the Chi site may be forward or reverse, and multiple Chi sites may be continuous or discontinuous. Depending on the target cell type, different Chi sites can be selected. In the same type of target cells, different intensity Chi site sequences can also be used.
  • Figure 5 is a schematic representation of gene editing in E. coli using the method of the present invention.
  • the target DNA fragment that is, the nucleic acid fragment constructed by the present invention
  • E. coli competent cells E. coli competent cells
  • the Escherichia coli recombinant cell containing the cat-sacB bidirectional screening marker gene is selected on the chloramphenicol-containing plate, and the selected recombinant cells are selected.
  • First cultured on normal LB medium without positive or negative screening then cultured in LB medium containing negative screening sucrose, and then screened for positive clones by LB plate containing sucrose. Finally, positive clones can be verified by PCR. .
  • Figures 6, 7 and 8 are maps of plasmids used in the examples of the present invention.
  • the method of the present invention can be used for editing genes at any position on the genome of prokaryotic cells such as E. coli or eukaryotic cells such as yeast, and can also be used for gene editing on plasmids or artificial chromosomes.
  • Example 1 Escherichia coli fadE was deleted without a trace
  • the tesCII fragment was obtained by reverse amplification using the upstream and downstream primers tesCp3 (AAAATTGCCACTATGCAAATTAATTACAGGG, SEQ ID NO.: 6) and tesCp4 (ACGTTTTGTGGTGCCGGATGCTC, SEQ ID NO.: 7).
  • the tesC IV fragment was obtained by PCR using pMD-tesC-cat-sacB as a template and amplification using the upstream and downstream primers tesCp1 and tesCp2.
  • the above culture medium was appropriately diluted with LB solid medium coated with 10% sucrose, and a single colony was grown, and tesCp1 and tesCp2 were used as primers for PCR detection, and the deletion-free strain was obtained by sequencing.
  • the FARS I fragment was obtained using the primers FARF (ATGGCAATACAGCAGGTACATCACG, SEQ ID NO.: 10) and FARR (TCAGGCAGCTTTTTTGCGCTG, SEQ ID NO.: 11) using the marine bacillus genome as a template.
  • the FARS II fragment was amplified by using pMD-tesC-cat-sacB as a template and tesCp4 and tesCp6 as primers.
  • the FARS III fragment was obtained by PCR using pMD-tesC-cat-sacB-FAR as a template and amplification using the upstream and downstream primers tesCp1 and tesCp2.
  • the above culture medium was appropriately diluted with LB solid medium coated with 10% sucrose, and a single colony was grown, and tesCp1 and tesCp2 were used as primers for PCR detection, and sequencing was carried out to obtain a seamless replacement strain.
  • Example 4 The Agrobacterium FAR gene was inserted into the downstream of the E. coli ldhA promoter without any trace
  • the upstream and downstream primers FARIp1 GCCGAATATCATGGTGGAAAATGG, SEQ ID NO.: 12
  • FARIp2 CGGCGATTGCTCCGTCTGC, SEQ ID NO.: 13
  • the partial sequence obtained the KldhAI fragment, which was obtained with pMD18-T to obtain pMD18-KldhAI.
  • FARIII fragment was obtained by PCR amplification using pMD18-KldhAI as a template and reverse amplification using the upstream and downstream primers FARIp3 (CATATGAATATCCTCCTTAGTTCCTATTCC, SEQ ID NO.: 14) and FARIp4 (AAGACTTTCTCCAGTGATGTTGAATCACAT, SEQ ID NO.: 15).
  • the FIR was obtained by amplifying the phosphorylation upstream and downstream primers P5 and FARIp6 ( CATATGAATATCCTCCTTAGTTCCTATTCCGAAGTTCCTATTCTCTAGAA ATCAAAGGGAAAACTGTCCATATGC, SEQ ID NO.:16, the underlined part is the homologous sequence upstream of the insertion site).
  • the FARI IV fragment was obtained by PCR using pMD-ldhAI-cat-sacB as a template and amplification using upstream and downstream primers FARIp3 and p5.
  • the FARI V fragment was obtained using the marine bacillus genome as a template.
  • the FARI VI fragment was obtained by PCR amplification using pMD-ldhAI-cat-chi-sacB-FAR as a template and FARIp1 and FARIp2 amplification.
  • the above culture medium was appropriately diluted with LB solid medium coated with 10% sucrose, and a single colony was grown, and FARIp1 and FARIp2 were used as primers for PCR detection, and the seamless insertion strain was obtained by sequencing.
  • the chisacB fragment was obtained by PCR using pEASY-cat-sacB as a template using phosphorylated upstream and downstream primers P5 (GTGACGGAAGATCACTTCGCAGA) and pccs-p6 (ATCAAAGGGAAAACTGTCCATATGC, SEQ ID NO.: 21).
  • Example 6 Efficient method for the deletion of E. coli fadE without any trace
  • fadE II A single-stranded DNA containing a homologous region at both ends of fadE, a 5'-end parallel repeat and cat-chi-sacB was synthesized and designated as fadE II (SEQ ID NO.: 22).
  • the above culture medium was appropriately diluted with LB solid medium coated with 10% sucrose, and a single colony was grown, and fadEp1 and fadEp2 were used as primers for PCR detection, and the deletion-free strain was obtained by sequencing.
  • the tesCI fragment was amplified by using the upstream and downstream primers tesCp1 and tesCp2 to amplify the tesC gene and the upstream and downstream partial sequences, and the fragment was pMD18-tesC with pMD18-T.
  • the tesC II fragment was obtained by PCR amplification using pMD18-tesC as a template and reverse amplification using the upstream and downstream primers tesCp3 and tesCp4.
  • the tesC III fragment was obtained by PCR using pMD-cat-chi-sacB as a template and amplification using phosphorylation upstream and downstream primers P5 and tesCp6.
  • the tesC IV fragment was obtained by PCR using pMD-tesC-cat-chi-sacB as a template and amplification using the upstream and downstream primers tesCp1 and tesCp2.
  • the above culture medium was appropriately diluted with LB solid medium coated with 10% sucrose, and a single colony was grown, and tesCp1 and tesCp2 were used as primers for PCR detection, and the deletion-free strain was obtained by sequencing.
  • Example 8 High-efficiency method of marine bacterium FAR gene for seamless replacement of Escherichia coli tesC
  • the FARSI fragment was obtained using the marine bacillus genome as a template.
  • the FARSII fragment was amplified by amplification with the primers tesCp4 and sacBR (ATCAAAGGGAAAACTGTCCATATGCAC, SEQ ID NO.: 23).
  • the FARSIII fragment was obtained by PCR using pMD-tesC-cat-chi-sacB-FAR as a template and amplification using the upstream and downstream primers tesCp1 and tesCp2.
  • the above culture medium was appropriately diluted with LB solid medium coated with 10% sucrose, and a single colony was grown, and tesCp1 and tesCp2 were used as primers for PCR detection, and sequencing was carried out to obtain a seamless replacement strain.
  • Example 9 High-efficiency method of F. transgenic FAR gene was inserted into the downstream of E. coli ldhA promoter
  • the FARIII fragment was obtained by PCR amplification using pMD18-KldhAI as a template and reverse amplification using the upstream and downstream primers FARIp3 and FARIp4.
  • the FARIIV fragment was obtained by PCR using pMD-ldhAI-cat-chi-sacB as a template and amplification using upstream and downstream primers FARIp3 and p5.
  • the FARIV fragment was obtained using the marine bacillus genome as a template.
  • the FARI VI fragment was obtained by PCR amplification using pMD-ldhAI-cat-chi-sacB-FAR as a template and FARIp1 and FARIp2 amplification.
  • Fragment FARSVI electroporates E. coli MG1655 competent cells containing pKD46, and is incubated with LB solid medium containing chloramphenicol after incubation. After the PCR was detected, the FAR insertion strain MGKLFARI was obtained by sequencing.
  • the above culture medium was appropriately diluted with LB solid medium coated with 10% sucrose, and a single colony was grown, and PCR was carried out by using tFARIp1 and FARIp2 as primers, and the seamless insertion strain was obtained by sequencing.

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Abstract

一种简便高效的基因编辑方法。该方法的特征是利用复制过程中基因组高频自发断裂和chi位点促进同源重组的原理，设计合成或构建带有并行重复序列和双向筛选标记基因的同源重组核酸片段，该片段在引入基因组后通过分子内同源重组消除引入的外源标记基因。相比需要进行两次转化的传统基因编辑方法，所述方法仅需要一次转化，可有效提高负筛选的效率，最高可达到50%。利用该方法可以实现基因的删除、替换、插入等基因编辑操作。

Description

一种简便高效的基因编辑方法 技术领域
本发明属于基因工程领域，具体地涉及一种基因编辑方法。
背景技术
近年来，随着第二代和第三代高通量测序技术的发展，功能基因组学和合成生物学的迅速发展迫切需要一种快速、无痕、高效的基因编辑工具，实现对基因组的定向编辑改造。模式微生物例如酵母菌、大肠杆菌等的无痕编辑技术近年来受到重视(Bird et al.,High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes.Nature methods,2012.9(1):p.103-109)，所开发的无痕编辑工具丰富多样。例如在酵母菌的双向筛选标记基因URA3的上游引入酶切位点提高无痕编辑的效率(Noskov,etal.,Tandem repeat coupled with endonuclease cleavage(TREC):a seamless modification tool for genome engineering in yeast.Nucleic acids research,2010.38(8):p.2570-2576)，在大肠杆菌开发多种双向筛选标记基因galK,thyA,tolC,tetA-sacB(Warming et al.,Simple and highly efficient BAC recombineering using galK selection.Nucleic acids research,2005.33(4):p.e36；Wong et al.,Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in Escherichia coli.Nucleic acids research,2005.33(6):p.e59；Gregg et al.,Rational optimization of tolC as a powerful dual selectable marker for genome engineering.Nucleic acids research,2014.DOI:10.1093/nar/gkt1374；Lietal.,Positive and negative selection using the tetA-sacB cassette:recombineering and P1transduction in Escherichia coli.Nucleic acids research,2013.41(22):p.e204)。但是，到目前为止基因编辑技术的难点，即双向筛选系统中标记基因的高效删除，仍未有效解决。
出于转基因的安全性及无痕编辑等要求，通常需要在完成所需的基因编辑后删除标记基因。传统的双向筛选标记基因的删除首先通过将含有插入位点上下游同源区的单链或双链核酸片段转化进入细胞，然后通过同源重组替换基因组上的抗性标记基因，最后通过在培养基中加入负筛底物完成对重组细胞的富集，以挑选出去除筛选标记基因，获得无痕编辑的细胞。因此，传统的基因编辑技术需要两次转化，第一次转化用于将带有目的基因和标记基因的核酸片段转入细胞进行同源重组，同源重组后目的基因和标记基因进入靶基因组；第二次转化用于删除标记基因，即将含有插入位点上下游同源区的核酸片段转入细胞，通过同源重组替换基因组上的抗性标记基因。此外，用这种方法删除标记基因的效率往往很低。
基因编辑技术中用于筛选重组子的双向筛选系统包含正筛选和负筛选，正筛选一般通过抗生素抗性标记基因赋予重组子对抗生素的抗性或通过营养缺陷型基因赋予在相应选择缺陷型培养基生长的特性来实现，负筛选一般利用某些基因赋予重组子对某些物质的敏感性来实现，例如利用sacB-neo融合基因来进行正/负筛选，neo(卡那霉素)抗性用于正筛选，而 由于sacB基因的表达而产生的蔗糖毒性用于负筛选(Warming et al.,Simple and highly efficient BAC recombineering using galK selection.Nucleic acids research,2005.33(4):p.e36)。双向筛选也可通过一个双向筛选标记基因来实现，例如tolC基因既可用于正筛选又可用于负筛选(Gregg et al.,Rational optimization of tolC as a powerful dual selectable marker for genome engineering.Nucleic acids research,2014.DOI:10.1093/nar/gkt1374)。负筛的成功率往往极低甚至常常失败。这主要是由于负筛选的致死过程需要有毒物质的生产与积累，在积累过程中细胞容易发生自发突变产生对有毒物质的抗性,使得细胞能够在保有负筛基因的情况下生存。不仅如此，负筛前的转化过程也常常引入对负筛物质不敏感的外源细胞而导致负筛过程的失败。
因此，本领域需要一种能够快速、高效地进行基因编辑的方法。
发明内容
针对传统基因编辑方法的上述及其他缺陷，本发明提供一种基因编辑方法，包括以下步骤：
a.提供靶细胞，所述靶细胞包含带有待修饰位点的靶核酸；
b.用包含所述待修饰位点的上下游同源序列、目的基因、并行重复序列和双向筛选标记基因的核酸片段转化所述靶细胞，以使所述核酸片段和靶核酸发生同源重组，从而在所述待修饰位点产生所需要的基因编辑；
c.进行正筛选以筛选出在靶核酸上整合了目的基因和双向筛选标记基因的重组子细胞；
d.允许所述核酸片段上的并行重复序列和待修饰位点的上游或下游的相应并行重复序列发生同源重组，以删除标记基因；
e.进行负筛选以富集删除了双向筛选标记基因的重组子细胞。
一种优选实施方式中，上述步骤b中核酸片段上的并行重复序列为靶核酸上待修饰位点上游或下游的原始序列。另一种优选的实施方式中，步骤b中核酸片段上的并行重复序列与靶核酸上紧邻修饰位点的上游或下游的序列同源，以实现无痕编辑。
另外一种实施方式中，步骤d中在不含正负筛选物质的培养基中培养步骤c筛选出的重组子细胞以允许所述核酸片段上的并行重复序列和待修饰位点的上游或下游的相应并行重复序列发生同源重组，从而删除标记基因。
另外一种实施方式中，步骤b中的双向筛选标记基因包含一个或多个Chi位点以提高双向筛选标记基因删除的效率。优选实施方式中，可以在不影响双向筛选标记基因功能的前提下，将Chi位点引入到双向筛选标记基因的任意一个或多个位点。所述Chi位点可以是正向的或反向的，多个Chi位点之间可以是连续的或不连续的。在不同类型的基因组中可使用不同的Chi位点，且在同一类型的基因组中可使用不同强度的Chi位点序列。
一种实施方式中，本发明方法用于原核细胞或真核细胞基因组任意位置基因的编辑，即靶核酸为原核细胞或真核细胞的基因组。另外一种实施方式中，本发明方法用于在质粒或人工染色体(BAC)上进行基因编辑，即靶核酸为质粒或人工染色体。
本发明的一个具体实施方式中还提供了一种用于同源重组的人工序列，所述序列为SEQ ID NO.:1、SEQ ID NO.:22、SEQ ID NO.:24、SEQ ID NO.:27或SEQ ID NO.:28。
本发明的另一个具体实施方式中还提供了一种Chi位点用于提高双向筛选标记基因删除的效率的应用。
本发明的另一个具体实施方式中还提供了一种同源重组的方法，包括如下步骤：a.提供靶细胞E.coli MG1655，所述靶细胞包含带有待修饰位点的靶核酸fadE基因；b.用包含fadE两端同源区，5’端并行重复序列和cat-sacB的fadE I基因，其序列为SEQ ID NO.:1，转化含pKD46的E.coli MG1655感受态靶细胞，以使所述核酸fadEI基因和靶核酸fadE发生同源重组；c.通过孵育后涂布于含氯霉素的LB固体培养基进行正筛选，获得重组菌MG1655ΔfadE::cat-sacB；d.纯化MG1655ΔfadE::cat-sacB并通过含10％蔗糖的LB液体培养基进行负筛选；e.通过PCR引物进行PCR检测，测序验证获得无痕删除菌株。
本发明的再一个具体实施方式中还提供了一种同源重组的方法，包括如下步骤：a.提供靶细胞E.coli MG1655，所述靶细胞包含待删除靶核酸tesC基因；b.利用PCR反应以E.coli MG1655基因组为模板，使用上下游引物，序列为SEQIDNO.:4的tesCp1和序列为SEQ ID NO.:5的tesCp2扩增大肠杆菌tesC基因及上下游部分序列获得tesC I片段，将该片段与pMD18-T获得pMD18-tesC；c.利用PCR反应以pMD18-tesC为模板，使用使用上下游引物，序列为SEQ ID NO.:6的tesCp3和序列为SEQ ID NO.:7的tesCp4反向扩增获得tesC II片段；d.利用PCR反应以pMD-cat-chi-sacB为模板，使用磷酸化上下游引物，序列为SEQ ID NO.:8的P5和序列为SEQ ID NO.:9的tesCp6扩增获得tesC III片段；e.连接片段tesC II和tesC III，转化E.coli DH5α菌株，获得pMD-tesC-cat-chi-sacB质粒；f.利用PCR反应以pMD-tesC-cat-chi-sacB为模板，使用上下游引物，序列为SEQ ID NO.:4的tesCp1和序列为SEQ ID NO.:5的tesCp2扩增获得tesC IV片段；g.片段tesC IV电转化含pKD46的E.coli MG1655感受态细胞，通过孵育后涂布于含氯霉素的LB固体培养基进行正筛选，获得重组菌MG tesC::cat-chi-sacB；h.纯化MG tesC::cat-chi-sacB并通过含10％蔗糖的LB液体培养基进行负筛选；i.以序列为SEQ ID NO.:4的tesCp1和序列为SEQ ID NO.:5的tesCp2为引物PCR检测，测序验证获得无痕删除菌株。
附图说明
附图1：基因编辑的三种主要方式。
附图2：并行重复序列辅助基因编辑的原理示意图。
附图3：含有Chi位点的双向筛选标记基因示意图。
附图4：并行重复序列和Chi位点辅助基因编辑的原理示意图。
附图5：用本发明方法在大肠杆菌中进行基因编辑的示意图。
附图6：质粒pMD18-T图谱(购自TaKaRa公司)。
附图7：质粒pEASY-cat-sacB图谱。
附图8：质粒pKD46图谱。
附图9：FADEI的序列，分别示出了FADE上游同源区、cat-sacB、FADE上游同源区、和FADE下游同源区。
附图10：FAA-URA3的序列，分别示出了faa1起始密码子上游同源区、faa1起始密码子上游同源区、faa1终止密码子下游同源区。
附图11：pMD-ldhAI-cat-sacB-FAR的序列，分别示出了插入位点上游序列、cat-sacB、插入位点上游同源区、FAR、插入位点下游同源区。
附图12：pMD-tesC-cat-sacB-FAR的序列，分别示出了tesC上游同源区、cat-sacB、tesC上游同源区、FAR、tesC下游同源区。
具体实施方式
本发明利用基因组高频自发断裂后同源重组重启复制叉的原理，通过在标记基因两端串联并行重复序列的方法，实现了一步转化两步重组完成基因编辑的过程。相对于传统的基因编辑方法(需要两次转化)，由于本发明提供的方法只需要一次转化就可以实现预期的改造和双向筛选标记基因的删除，因此在很大程度上简化了基因编辑的操作步骤。另外，本发明提供的方法提高了双向筛选标记基因的删除效率。不受任何理论的限制，双向筛选标记基因删除效率的提高可能是由于传统上删除双向筛选标记基因的方法是通过分子间的同源重组，这种重组在很大程度上受到转化效率(一般不超过1/1000)的影响，而本发明的方法是靠分子内重组完成的，每个细胞都含有发生同源重组的片段。此外，本发明方法通过在双向筛选标记基因中引入Chi位点的方式提高了负筛选的效率，最高可达到50％。由于本发明的方法可以进行基因的无痕编辑，因此可以连续实现基因的删除、替换、插入等操作。
在本发明上下文中，术语“靶细胞”是指待转化的细胞，靶细胞中含有靶核酸，用根据本发明设计和构建的核酸片段转化该细胞，使核酸片段与靶核酸发生同源重组，从而实现所需的基因编辑。
术语“靶核酸”是指需要进行基因编辑的核酸，靶核酸可以是原核细胞或真核细胞的基因组，也可以是质粒或人工染色体(BAC)等。
本发明上下文中，术语“核酸”包括DNA、cDNA、和RNA。
术语“基因编辑”是指在待修饰位点改变基因序列，包括基因删除、插入和替换。本发明上下文中的基因编辑包括对基因或任意碱基进行编辑，即用基因或任意碱基删除、插入或替换靶核酸上原始的基因或碱基。
术语“无痕编辑”或“无痕基因编辑”是指经编辑的核酸上不存在不需要的多余碱基。
术语“待修饰位点”或“修饰位点”是指靶核酸上需要进行基因编辑的具体位置。
术语“同源”，例如“同源序列”中的“同源”，是指两个核酸之间的核苷酸序列相似性或一致性。本领域技术人员知晓，可通过DNA-DNA或DNA-RNA杂交进行同源性的评估(如Haines和Higgins(主编)的《核酸杂交》(Nucleic Acid Hybridization)(IRL出版社，牛津，英国)中所述)，或通过比较两个核酸之间的序列一致性来进行同源性的评估。对本发明目的而言，“同源”是指两个核酸之间至少具有30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或100％的序列相似性或一致性，优选具有60％、70％、80％、90％、95％、96％、97％、98％、99％或100％的序列相似性或一致性，更优选具有80％、90％、95％、96％、97％、98％、99％或100％的序列相似性或一致性，最优选具有90％、95％、96％、97％、98％、 99％或100％的序列相似性或一致性。可用于进行序列比对的软件例如BLAST和ClustalW程序，两种程序都可以从网上找到。
术语“本发明构建的核酸片段”或“根据本发明设计和构建的核酸片段”是指包含待修饰位点的上下游同源序列、目的基因(当用于基因删除时，不存在目的基因)、并行重复序列和双向筛选标记基因的核酸片段。其中与待修饰位点的上下游序列同源的序列位于该核酸片段的两端，用于与靶核酸同源重组。
术语“目的基因”是指用于在待修饰位点进行基因编辑的基因，例如在待修饰位点取代靶核酸上原始的基因或插入到靶核酸上，当用于在靶核酸上删除基因或碱基时，不需要目的基因，即在本发明构建的核酸片段上目的基因为不存在。
术语“并行重复序列”是指在本发明构建的核酸片段上的一段序列与靶核酸上待修饰位点的上游或下游序列同源，例如本发明构建的核酸片段5’端含有与待修饰位点的下游(即3’端)同源的并行重复序列(即待修饰位点的下游的相应并行重复序列)，或本发明构建的核酸片段3’端含有与待修饰位点的上游(即5’端)同源的并行重复序列(即待修饰位点的上游的相应并行重复序列)。本发明构建的核酸片段上的这段序列和靶核酸上与其同源的序列在本发明中均称为“并行重复序列”。并行重复序列的长度可变，例如在30个碱基至1000个碱基范围内，本领域技术人员可根据具体需要确定并行重复序列的长度。术语“双向筛选标记基因”或“标记基因”是指既可以进行正筛选又可以进行负筛选的基因，可以是一个或多个基因，例如，tolC、galK、thyA、neo-sacB、tetA-sacB、cat-sacB等。
“正筛选”具有本领域技术人员通常理解的含义，即一般通过抗生素抗性标记基因赋予重组子对抗生素的抗性或通过营养缺陷型基因赋予在相应选择缺陷型培养基生长的特性来实现，例如利用正筛选物卡那霉素或氯霉素，尿嘧啶缺陷型培养基等。
“负筛选”具有本领域技术人员通常理解的含义，即其特征一般是在特定的限定条件下，负筛选物的存在对细胞有不利影响，例如使细胞死亡或阻碍/抑制细胞生长。最常用的负筛选标记是枯草芽孢杆菌(Bacillus subtilis)的sacB。当sacB被引入异源宿主例如大肠杆菌时，其在外源蔗糖存在的情况下会导致细胞死亡。
“Chi位点”或“Chi序列”是基因组中一段短的DNA片段，在该位点附近发生同源重组的几率增加。不同类型有机体的基因组中Chi位点的序列可能是不同的。
下面参考附图对本发明做进一步的说明。
图1示出了基因编辑的三种主要方式，即基因或碱基的删除、替换或插入。
图2是并行重复序列辅助基因编辑的原理示意图。如图中所示，根据本发明设计和构建的核酸片段在其两端含有与待修饰位点的上游和下游同源的序列，因此所述片段在转化到细胞中之后能够与靶核酸(基因组或质粒)发生同源重组，所述核酸片段整合到靶核酸上，将待修饰位点的基因或碱基删除、替换待修饰位点的基因或碱基、或者插入到待修饰位点。在发生上述第一步的同源重组之后，通过正筛选富集重组子，例如如果所述核酸片段上的双向筛选标记基因之一是cat，即氯霉素抗性基因，则可以用含有氯霉素的培养基筛选经过转化的细胞，以富集发生了同源重组从而具有氯霉素抗性的重组子细胞。
在正筛选之后，需要去除标记基因。本发明方法利用两个并行重复序列之间的同源重组 剔除标记基因。所述并行重复序列一个位于待筛选位点的上游或下游，一个在根据本发明构建的核酸片段上，两个并行重复序列之间是双向筛选标记基因。如果本发明构建的核酸片段在5’端含有并行重复序列，则该序列与待修饰位点的3’端上游序列同源，如果本发明构建的核酸片段在3’端含有并行重复序列，则该序列与待修饰位点的5’端上游序列同源，从而当这两个并行重复序列位于同一分子内时能够发生同源重组并将它们之间的标记基因删除。所述两个并行重复序列之间至少具有30％、40％、50％、60％、70％、80％、90％、95％、96％、97％、98％、99％或100％的序列相似性或一致性，优选具有60％、70％、80％、90％、95％、96％、97％、98％、99％或100％的序列相似性或一致性，更优选具有80％、90％、95％、96％、97％、98％、99％或100％的序列相似性或一致性，最优选具有90％、95％、96％、97％、98％、99％或100％的序列相似性或一致性。一种特别优选的实施方式中，核酸片段上的并行重复序列为靶核酸上待修饰位点上游或下游的原始序列。并行重复序列的长度可根据需要变化，例如在30至1000个碱基范围内，本领域技术人员可根据具体需要确定并行重复序列的长度。如图2所示，在发生第一步同源重组之后，靶核酸上含有这两个并行重复序列，两个并行重复序列之间隔着标记基因，然后允许这两个并行重复序列发生分子内同源重组，例如通过在不含正负筛选物质的培养基中培养重组子细胞一段时间，从而除去标记基因。
本发明方法可以进行基因的无痕编辑。仍然参考图2，如果本发明构建的核酸片段上的并行重复序列与靶核酸上紧邻修饰位点的上游或下游的序列同源，则可以实现无痕编辑。“紧邻修饰位点的上游或下游的序列”是指所述上游或下游序列与修饰位点之间是连续的，没有碱基间隔。
本发明方法中，在双向筛选标记基因中引入一个或多个Chi位点进一步提高了双向筛选标记基因删除的效率(参见图3和图4)，使最终负筛选的效率最高可达到50％。可以在不影响双向筛选标记基因功能的前提下，将Chi位点引入到双向筛选标记基因的任意一个或多个位点。所述Chi位点可以是正向的或反向的，多个Chi位点之间可以是连续的或不连续的。根据不同的靶细胞类型，可选择使用不同的Chi位点，在同一类型靶细胞中，也可选择使用不同强度的Chi位点序列。
图5是用本发明方法在大肠杆菌中进行基因编辑的示意图。首先用目标DNA片段即本发明构建的核酸片段转化大肠杆菌感受态细胞，然后在含有氯霉素的平板上筛选含有cat-sacB双向筛选标记基因的大肠杆菌重组子细胞，筛选出的重组子细胞先在不含正负筛选物的普通LB培养基上培养，然后用含有负筛选物蔗糖的LB培养基培养，再用含有蔗糖的LB平板筛选富集阳性克隆，最后可用PCR验证获得的阳性克隆。
图6、图7和图8是本发明实施例中使用的质粒的图谱。
本发明方法可用于原核细胞例如大肠杆菌或真核细胞例如酵母的基因组上任意位置基因的编辑，也可用于在质粒或人工染色体上进行基因编辑。
上文结合附图对本发明做了详细的描述。本领域技术人员理解，这些描述只是为了更好地阐释和理解本发明，并不意于对本发明的范围做任何限制。下文将结合具体实施例对本发明作进一步的说明。同样，这些实施例只是为了使本领域技术人员能够更好地理解本发明，并不构成对本发明范围的任何限制。本发明的范围仅由所附的权利要求书限定。
本领域技术人员认可，可对本发明的形式进行多种变化和改动而不脱离本发明的精神和范围，这些变化和改动由于属于本发明的等同形式而落入本发明权利要求保护的范围内。
除本文另有说明以外，本申请中所用术语具有本领域技术人员所理解的一般含义。分子生物学和基因工程领域的实验操作，例如质粒构建、转化和筛选的一般技术是本领域技术人员熟知的，也可参见本领域的参考书例如《分子克隆实验指南》，第3版，J.萨姆布鲁克。大肠杆菌(Escherichia coli)的基因组序列可在HTTP://WWW.NCBI.NLM.NIH.GOV/GENOME/167？PROJECT_ID＝57779获得。海洋杆菌(Marinobacter aquaeolei VT8)的基因组序列可在HTTP://WWW.NCBI.NLM.NIH.GOV/GENOME/？TERM＝MARINOBACTER+AQUAEOLEI获得。
实施例
实施例1无痕删除大肠杆菌(Escherichia coli)fadE
(1)合成含有fadE两端同源区，5’端并行重复序列和cat-sacB的DNA，命名为fadEI(SEQ ID NO.:1，也参见图9)。
(2)片段fadEI转化含pKD46的E.coli MG1655感受态细胞，孵育后涂布于含氯霉素的LB固体培养基。待长出后经PCR和测序验证获得重组菌MG1655ΔfadE::cat-sacB。
(3)纯化MG1655ΔfadE::cat-sacB，如附图5所示负筛选流程，取单菌落接种于100mLLB液体培养基，培养12h；取1mL培养液接种于含10％蔗糖的LB液体培养基，培养12h；取300μL培养液接种于含10％蔗糖的LB液体培养基，培养12h。
(4)取上述培养液适当稀释涂布10％蔗糖的LB固体培养基，待长出单菌落，以fadEp1(TTGAAACCGAAATCATTACCGACGC，SEQ ID NO.:2)和fadEp2(CGTGTTATCGCCAGGCTTTAGGAGG，SEQ ID NO.:3)为引物PCR进行PCR检测，PCR反应程序为：94℃4min
94℃30s，62℃30s，72℃2min35cycles，
72℃5min(以下所有实施例所用的PCR程序均与此处相同)
测序验证获得无痕删除菌株。
实施例2无痕删除大肠杆菌tesC
(1)利用PCR反应以E.coli MG1655基因组为模板，使用上下游引物tesCp1(GCACTGCTCATTACCCTGTCCCTG，SEQ ID NO.:4)和tesCp2(TGGATGTCACCCTGCTCAACGAG，SEQIDNO.:5)扩增大肠杆菌tesC基因及上下游部分序列获得tesCI片段，将该片段与pMD18-T获得pMD18-tesC。
(2)利用PCR反应以pMD18-tesC为模板，使用上下游引物tesCp3(AAAATTGCCACTATGCAAATTAATTACAGGG，SEQ ID NO.:6)和tesCp4(ACGTTTTGTGGTGCCGGATGCTC，SEQ ID NO.:7)反向扩增获得tesCII片段。
(3)利用PCR反应以pEASY-cat-sacB为模板，使用磷酸化上下游引物P5 (GTGACGGAAGATCACTTCGCAGA，SEQ ID NO.:8)和tesCp6(AAAATTGCCACTATGCAAATTAATTACAGGGTTAATACCGCCAGATTACGATCAAAGGGAAAACTGTCCATATGC，SEQ ID NO.:9，下划线部分为tesC基因上游同源序列)扩增获得tesC III片段。
(4)连接片段tesC II和tesC III，转化E.coli DH5α菌株，获得pMD-tesC-cat-sacB质粒。
(5)利用PCR反应以pMD-tesC-cat-sacB为模板，使用上下游引物tesCp1和tesCp2扩增获得tesC IV片段。
(6)片段tesC IV电转化含pKD46的E.coli MG1655感受态细胞，孵育后涂布与含氯霉素的LB固体培养基。待长出后PCR检测，测序验证获得重组菌MGΔtesC::cat-sacB。
(7)纯化MGΔtesC::cat-sacB，如附图5所示负筛选流程，取单菌落接种于100mLLB液体培养基，培养12h；取1mL培养液接种于含10％蔗糖的LB液体培养基，培养12h：取300μL培养液接种于含10％蔗糖的LB液体培养基，培养12h。
(8)取上述培养液适当稀释涂布10％蔗糖的LB固体培养基，待长出单菌落，以tesCp1和tesCp2为引物PCR检测，测序验证获得无痕删除菌株。
实施例3海洋杆菌(Marinobacter aquaeolei VT8)FAR基因无痕替换大肠杆菌tesC
(1)使用引物FARF(ATGGCAATACAGCAGGTACATCACG，SEQ ID NO.:10)和FARR(TCAGGCAGCTTTTTTGCGCTG，SEQ ID NO.:11)，以海洋杆菌基因组为模板获得FARS I片段。
(2)以pMD-tesC-cat-sacB为模板，以tesCp4和tesCp6为引物扩增获得FARS II片段。
(3)连接片段FARSI和FARSII，转化E.coliDH5α菌株，获得pMD-tesC-cat-sacB-FAR质粒(SEQ ID NO.:27，也参见附图12)。
(4)利用PCR反应以pMD-tesC-cat-sacB-FAR为模板，使用上下游引物tesCp1和tesCp2扩增获得FARS III片段。
(5)片段FARS III电转化含pKD46的E.coli MG1655感受态细胞，孵育后涂布与含氯霉素的LB固体培养基。待长出后PCR检测，测序验证获得重组菌MGΔtesC::cat-sacB-FAR。
(6)纯化MGΔtesC::cat-sacB-FAR，如附图5所示负筛选流程，取单菌落接种于100mL LB液体培养基，培养12h；取1mL培养液接种于含10％蔗糖的LB液体培养基，培养12h：取300μL培养液接种于含10％蔗糖的LB液体培养基，培养12h。
(7)取上述培养液适当稀释涂布10％蔗糖的LB固体培养基，待长出单菌落，以tesCp1和tesCp2为引物PCR检测，测序验证获得无痕替换菌株。
实施例4海洋杆菌FAR基因无痕插入大肠杆菌ldhA启动子下游
(1)利用PCR反应以E.coliMG1655基因组为模板，使用上下游引物FARIp1(GCCGAATATCATGGTGGAAAATGG，SEQ ID NO.:12)和FARIp2(CTGGCGATTGCTCCGTCTGC，SEQ ID NO.:13)扩增大肠杆菌ldhA启动子上下游部分序列获得KldhAI片段，将该片段与pMD18-T获得pMD18-KldhAI。
(2)利用PCR反应以pMD18-KldhAI为模板，使用使用上下游引物FARIp3(CATATGAATATCCTCCTTAGTTCCTATTCC，SEQ ID NO.:14)和FARIp4(AAGACTTTCTCCAGTGATGTTGAATCACAT，SEQ ID NO.:15)反向扩增获得FARIII片段。
(3)利用PCR反应以pMD-cat-sacB为模板，使用磷酸化上下游引物P5和FARIp6(CATATGAATATCCTCCTTAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAATCAAAGGGAAAACTGTCCATATGC，SEQ ID NO.:16，下划线部分为插入位点上游同源序列)扩增获得FARI III片段
(4)连接片段FARI II和FARI III，转化E.coli DH5α菌株，获得pMD-ldhAI-cat-sacB质粒。
(5)利用PCR反应以pMD-ldhAI-cat-sacB为模板，使用上下游引物FARIp3和p5扩增获得FARI IV片段。
(6)使用磷酸化引物FARF和FARR，以海洋杆菌基因组为模板获得FARI V片段
(7)连接片段FARI IV和FARI V，转化E.coliDH5α菌株，获得pMD-ldhAI-cat-sacB-FAR(SEQ ID NO.:28，也参见附图11)质粒。
(8)利用PCR反应以pMD-ldhAI-cat-chi-sacB-FAR为模板，FARIp1和FARIp2扩增获得FARI VI片段。
(9)片段FARS VI电转化含pKD46的E.coli MG1655感受态细胞，孵育后涂布与含氯霉素的LB固体培养基。待长出后PCR检测，测序验证获得FAR插入菌株MGKLFARI。
(10)纯化MGKLFARI，如附图5所示负筛选流程，取单菌落接种于100mLLB液体培养基，培养12h；取1mL培养液接种于含10％蔗糖的LB液体培养基，培养12h：取300μL培养液接种于含10％蔗糖的LB液体培养基，培养12h。
(11)取上述培养液适当稀释涂布10％蔗糖的LB固体培养基，待长出单菌落，以FARIp1和FARIp2为引物PCR检测，测序验证获得无痕插入菌株。
实施例5pMD-cat-chi-sacB的构建
(1)利用PCR反应以pEASY-cat-sacB为模板使用上下游引物pccs-p1(GTGACGGAAGATCACTTCGCAGA，SEQ ID NO.:17)和pccs-p2(CCACCAGCCAGTAACAAACCCGCGCGATTT，SEQ ID NO.:18，下划线为chi位点序列)获得catchi片段，将该片段与pMD18-T获得pMD18-cat-chi。
(2)利用PCR反应以pMD18-cat-chi为模板使用上下游引物pccs-P3(CCACCAGCCAGTAACAAACCCG，SEQIDNO.:19，下划线为chi位点序列)和pccs-P4(ATCTCTAGAGGATCCCCGGGTACC，SEQ ID NO.:20)获得pMD18-catchiF片段。
(3)利用PCR反应以pEASY-cat-sacB为模板使用磷酸化的上下游引物P5(GTGACGGAAGATCACTTCGCAGA)和pccs-p6(ATCAAAGGGAAAACTGTCCATATGC，SEQ ID NO.:21)获得chisacB片段。
(4)连接chisacB片段和pMD18-catchiF片段，转化E.coli DH5α菌株，获得pMD-cat-chi-sacB质粒。
实施例6高效法无痕删除大肠杆菌fadE
(1)合成含有fadE两端同源区，5’端并行重复序列和cat-chi-sacB的单链DNA，命名为fadE II(SEQ ID NO.:22)。
(2)片段fadE II电转化含pKD46的E.coli MG1655感受态细胞，孵育后涂布与含氯霉素的LB固体培养基。待长出后PCR检测，测序验证获得重组菌MGΔfadE::cat-chi-sacB
(3)纯化MGΔfadE::cat-chi-sacB，如附图5所示负筛选流程，取单菌落接种于100mL LB液体培养基，培养12h；取1mL培养液接种于含10％蔗糖的LB液体培养基，培养12h：取300μL培养液接种于含10％蔗糖的LB液体培养基，培养12h。
(4)取上述培养液适当稀释涂布10％蔗糖的LB固体培养基，待长出单菌落，以fadEp1和fadEp2为引物PCR检测，测序验证获得无痕删除菌株。
实施例7高效法无痕删除大肠杆菌tesC
(1)利用PCR反应以E.coli MG1655基因组为模板，使用上下游引物tesCp1和tesCp2扩增大肠杆菌tesC基因及上下游部分序列获得tesCI片段，将该片段与pMD18-T获得pMD18-tesC。
(2)利用PCR反应以pMD18-tesC为模板，使用使用上下游引物tesCp3和tesCp4反向扩增获得tesC II片段。
(3)利用PCR反应以pMD-cat-chi-sacB为模板，使用磷酸化上下游引物P5和tesCp6扩增获得tesC III片段。
(4)连接片段tesC II和tesC III，转化E.coli DH5α菌株，获得pMD-tesC-cat-chi-sacB质粒。
(5)利用PCR反应以pMD-tesC-cat-chi-sacB为模板，使用上下游引物tesCp1和tesCp2扩增获得tesC IV片段。
(6)片段tesC IV电转化含pKD46的E.coli MG1655感受态细胞，孵育后涂布与含氯霉素的LB固体培养基。待长出后PCR检测，测序验证获得重组菌MGΔtesC::cat-chi-sacB。
(7)纯化MGΔtesC::cat-chi-sacB，如附图5所示负筛选流程，取单菌落接种于100mL LB液体培养基，培养12h；取1mL培养液接种于含10％蔗糖的LB液体培养基，培养12h：取300μL培养液接种于含10％蔗糖的LB液体培养基，培养12h。
(8)取上述培养液适当稀释涂布10％蔗糖的LB固体培养基，待长出单菌落，以tesCp1和tesCp2为引物PCR检测，测序验证获得无痕删除菌株。
实施例8高效法海洋杆菌FAR基因无痕替换大肠杆菌tesC
(1)使用引物FARF和FARR，以海洋杆菌基因组为模板获得FARSI片段。
(2)以pMD-tesC-cat-chi-sacB为模板，用引物tesCp4和sacBR(ATCAAAGGGAAAACTGTCCATATGCAC，SEQ ID NO.:23)扩增获得FARSII片段。
(3)连接片段FARSI和FARSII，转化E.coliDH5α菌株，获得pMD-tesC-cat-chi-sacB-FAR质粒。
(4)利用PCR反应以pMD—tesC-cat-chi-sacB-FAR为模板，使用上下游引物tesCp1和tesCp2扩增获得FARSIII片段。
(5)片段FARSIII电转化含pKD46的E.coliMG1655感受态细胞，孵育后涂布与含氯霉素的LB固体培养基。待长出后PCR检测，测序验证获得重组菌MGΔtesC::cat-chi-sacB-FAR。
(6)纯化MGΔtesC::cat-chi-sacB-FAR，如附图5所示负筛选流程，取单菌落接种于100mL LB液体培养基，培养12h；取1mL培养液接种于含10％蔗糖的LB液体培养基，培养12h：取300μL培养液接种于含10％蔗糖的LB液体培养基，培养12h。
(7)取上述培养液适当稀释涂布10％蔗糖的LB固体培养基，待长出单菌落，以tesCp1和tesCp2为引物PCR检测，测序验证获得无痕替换菌株。
实施例9高效法海洋杆菌FAR基因无痕插入大肠杆菌ldhA启动子下游
(1)利用PCR反应以E.coli MG1655基因组为模板，使用上下游引物FARIp1和FARIp2扩增大肠杆菌ldhA启动子上下游部分序列获得KldhAII片段，将该片段与pMD18-T获得pMD18-KldhAI。
(2)利用PCR反应以pMD18-KldhAI为模板，使用使用上下游引物FARIp3和FARIp4反向扩增获得FARIII片段。
(3)利用PCR反应以pMD-cat-chi-sacB为模板，使用磷酸化上下游引物P5和FARIp6扩增获得FARI III片段
(4)连接片段FARI II和FARI III，转化E.coli DH5α菌株，获得pMD-ldhAI-cat-chi-sacB质粒。
(5)利用PCR反应以pMD-ldhAI-cat-chi-sacB为模板，使用上下游引物FARIp3和p5扩增获得FARIIV片段。
(6)使用磷酸化引物FARF和FARR，以海洋杆菌基因组为模板获得FARIⅤ片段
(7)连接片段FARIIV和FARIV，转化E.coli DH5α菌株，获得pMD-ldhAI-cat-chi-sacB-FAR质粒。
(8)利用PCR反应以pMD-ldhAI-cat-chi-sacB-FAR为模板，FARIp1和FARIp2扩增获得FARI VI片段。
(9)片段FARSVI电转化含pKD46的E.coli MG1655感受态细胞，孵育后涂布与含氯霉素的LB固体培养基。待长出后PCR检测，测序验证获得FAR插入菌株MGKLFARI。
(10)纯化MGKLFARI，如附图5所示负筛选流程，取单菌落接种于100mL LB液体培养基，培养12h；取1mL培养液接种于含10％蔗糖的LB液体培养基，培养12h：取300μL培养液接种于含10％蔗糖的LB液体培养基，培养12h。
(11)取上述培养液适当稀释涂布10％蔗糖的LB固体培养基，待长出单菌落，以tFARIp1和FARIp2为引物PCR检测，测序验证获得无痕插入菌株。
实施例10无痕敲除酵母菌FAA1
(1)合成含有FAA1两端同源区，5’端并行重复序列和URA3的单链DNA，命名为FAA-URA3(SEQ ID NO.:24，也参见附图10)。
(2)片段FAA-URA3电转化酿酒酵母感受态细胞，孵育后涂布于SD培养基。待长出后PCR检测，测序验证获得FAA1敲除菌TAMkf。
(3)纯化TAMkf，取单菌落接种于SD+尿嘧啶液体培养基，培养24h；取1mL培养液接种于SD+尿嘧啶+5-FOA(即5-氟乳清酸)液体培养基，培养24h：取300μL培养液接种于SD+尿嘧啶+5-FOA液体培养基，培养24h。
(4)取上述培养液划线于SD+尿嘧啶+5-FOA固体培养基，培养50h，挑取单菌落后点对点培养于SD+尿嘧啶和SD固体培养基。
(5)选取在SD固体培养基不生长，在SD+尿嘧啶培养基生长的菌落，以FAAp1(TTAGGATACAATAAAAACTAGAACAAACAC,SEQ ID NO.:25)和FAAp2(CTATCATGGAAATGTTGATCC,SEQ ID NO.:26)为引物PCR，测序验证获得无痕删除菌株。

Claims (14)

  1. 一种基因编辑方法，包括以下步骤：
    a.提供靶细胞，所述靶细胞包含带有待修饰位点的靶核酸；
    b.用包含所述待修饰位点的上下游同源序列、目的基因、并行重复序列和双向筛选标记基因的核酸片段转化所述靶细胞，以使所述核酸片段和靶核酸发生同源重组，从而在所述待修饰位点产生所需要的基因编辑；
    c.进行正筛选以筛选出在靶核酸上整合了目的基因和双向筛选标记基因的重组子细胞；
    d.允许所述核酸片段上的并行重复序列和待修饰位点的上游或下游的相应并行重复序列发生同源重组，以删除标记基因；
    e.进行负筛选以富集删除了双向筛选标记基因的重组子细胞。
  2. 权利要求1的方法，其特征在于步骤b中核酸片段上的并行重复序列为靶核酸上待修饰位点上游或下游的原始序列。
  3. 权利要求1的方法，其特征在于步骤b中核酸片段上的并行重复序列与靶核酸上紧邻待修饰位点的上游或下游的序列同源，以实现无痕编辑。
  4. 权利要求1的方法，其特征在于步骤d中在不含正负筛选物质的培养基中培养步骤c筛选出的重组子细胞以允许所述核酸片段上的并行重复序列和待修饰位点的上游或下游的相应并行重复序列发生同源重组，从而删除标记基因。
  5. 权利要求1-4中任一项的方法，其特征在于步骤b中的双向筛选标记基因包含一个或多个Chi位点以提高双向筛选标记基因删除的效率。
  6. 权利要求5的方法，其特征在于在不影响双向筛选标记基因功能的前提下，将Chi位点引入到双向筛选标记基因的任意一个或多个位点。
  7. 权利要求5的方法，其特征在于所述Chi位点可以是正向的或反向的，多个Chi位点之间可以是连续的或不连续的。
  8. 权利要求5的方法，其特征在于在不同类型的基因组中可使用不同的Chi位点，在同一类型的基因组中可使用不同强度的Chi位点序列。
  9. 权利要求1的方法，其特征在于所述方法用于原核细胞或真核细胞基因组任意位置基因的编辑。
  10. 权利要求1的方法，其特征在于所述方法用于在质粒或人工染色体上进行基因编辑。
  11. 一种用于同源重组的人工序列，其特征在于，所述序列为SEQ ID NO.:1、SEQ ID NO.:22、SEQ ID NO.:24、SEQ ID NO.:27或SEQ ID NO.:28。
  12. 一种Chi位点的应用，其特征在于，将其用于提高双向筛选标记基因删除的效率。
  13. 一种同源重组的方法，其特征在于，无痕删除大肠杆菌的fadE基因，包括如下步骤：
    a.提供靶细胞E.coli MG1655，所述靶细胞包含带有待修饰位点的靶核酸fadE基因；
    b.用包含fadE两端同源区，5’端并行重复序列和cat-sacB的fadE I基因，其序列为SEQ ID NO.:1，转化含pKD46的E.coli MG1655感受态靶细胞，以使所述核酸fadE I基因和 靶核酸fadE发生同源重组；
    c.通过孵育后涂布于含氯霉素的LB固体培养基进行正筛选，获得重组菌MG1655ΔfadE::cat-sacB；
    d.纯化MG1655ΔfadE::cat-sacB并通过含10％蔗糖的LB液体培养基进行负筛选；
    e.通过PCR引物进行PCR检测，测序验证获得无痕删除菌株。
  14. 一种同源重组的方法，其特征在于，无痕删除大肠杆菌tesC基因，包括如下步骤：
    a.提供靶细胞E.coli MG1655，所述靶细胞包含待删除靶核酸tesC基因；
    b.利用PCR反应以E.coli MG1655基因组为模板，使用上下游引物，序列为SEQ ID NO.:4的tesCp1和序列为SEQ ID NO.:5的tesCp2扩增大肠杆菌tesC基因及上下游部分序列获得tesC I片段，将该片段与pMD18-T获得pMD18-tesC；
    c.利用PCR反应以pMD18-tesC为模板，使用使用上下游引物，序列为SEQ ID NO.:6的tesCp3和序列为SEQ ID NO.:7的tesCp4反向扩增获得tesCII片段；
    d.利用PCR反应以pMD-cat-chi-sacB为模板，使用磷酸化上下游引物，序列为SEQ ID NO.:8的P5和序列为SEQ ID NO.:9的tesCp6扩增获得tesC III片段；
    e.连接片段tesC II和tesC III，转化E.coli DH5α菌株，获得pMD-tesC-cat-chi-sacB质粒；
    f.利用PCR反应以pMD-tesC-cat-chi-sacB为模板，使用上下游引物，序列为SEQ ID NO.:4的tesCp1和序列为SEQ ID NO.:5的tesCp2扩增获得tesC IV片段；
    g.片段tesC IV电转化含pKD46的E.coli MG1655感受态细胞，通过孵育后涂布于含氯霉素的LB固体培养基进行正筛选，获得重组菌MG tesC::cat-chi-sacB；
    h.纯化MG tesC::cat-chi-sacB并通过含10％蔗糖的LB液体培养基进行负筛选；
    i.以序列为SEQ ID NO.:4的tesCp1和序列为SEQ ID NO.:5的tesCp2为引物PCR检测，测序验证获得无痕删除菌株。
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