CN103898140A - Simple efficient gene editing method - Google Patents
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Abstract
本发明公开了一种简便高效的基因编辑方法。本发明方法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。相对于需要进行两次转化的传统基因编辑方法，本发明方法仅需要一次转化，并且可有效地提高负筛选的效率，最高可达到50%。本发明的方法可以实现基因的删除、替换、插入等基因编辑操作。
The invention discloses a simple and efficient gene editing method. The method of the present invention utilizes the principle of high-frequency spontaneous fragmentation of the genome and the promotion of homologous recombination by the chi site during the replication process to design, synthesize or construct a homologous recombination nucleic acid fragment with parallel repeat sequences and bidirectional screening marker genes. After the fragment is introduced into the genome Elimination of introduced exogenous marker genes by intramolecular homologous recombination. Compared with the traditional gene editing method that requires two transformations, the method of the present invention only requires one transformation, and can effectively improve the efficiency of negative selection, up to 50%. The method of the present invention can realize gene editing operations such as gene deletion, replacement, and insertion.
Description
Technical field
The invention belongs to genetically engineered field, relate to particularly a kind of gene editing method.
Background technology
In recent years, along with the development of the s-generation and third generation high throughput sequencing technologies, developing rapidly in the urgent need to quick, seamless, the efficient gene editing instrument of one of functional genomics and synthetic biology, realizes genomic directed editor's transformation.The seamless editing technique of mode trickle biology such as yeast, intestinal bacteria etc. (the Bird et al. that comes into one's own in recent years, High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes.Nature methods, 2012.9 (1): p.103-109), the seamless edit tool of developing is rich and varied.For example introduce restriction enzyme site in the upstream of saccharomycetic bidirectional screening marker gene URA3 and improve seamless editor's efficiency (Noskov, et al., 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), develop multiple bidirectional screening marker gene galK intestinal bacteria, 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, Li et al., Positive and negative selection using the tetA-sacB cassette:recombineering and P1transduction in Escherichia coli.Nucleic acids research, 2013.41 (22): p.e204).But, the up to the present difficult point of gene editing technology, i.e. the efficient deletion of marker gene in bidirectional screening system, effectively solves not yet.
For requirements such as genetically modified security and seamless editors, conventionally delete flag gene after required gene editing need to be completed.The deletion of traditional bidirectional screening marker gene is first by entering cell by the strand that contains insertion point upstream and downstream homologous region or double stranded nucleic acid fragment conversion, then replace the resistant maker gene on genome by homologous recombination, finally by adding negative sieve substrate to complete the enrichment to reconstitution cell in substratum, remove selection markers gene to pick out, obtain seamless editor's cell.Therefore, traditional twice conversion of gene editing Technology Need, transforms for the first time for carrying out homologous recombination by proceeding to cell with the nucleic acid fragment of goal gene and marker gene, and after homologous recombination, goal gene and marker gene enter target gene group; Transform for delete flag gene for the second time, the nucleic acid fragment that is about to contain insertion point upstream and downstream homologous region proceeds to cell, replaces the resistant maker gene on genome by homologous recombination.In addition, the efficiency of delete flag gene is often very low in this way.
In gene editing technology, comprise and just screen and bear screening for screening the bidirectional screening system of recon, just screening generally and giving recon to antibiotic resistance or give the characteristic of growing at corresponding selection defective type substratum by auxotroph gene and realize by antibiotics resistance marker gene, negative some gene of screening general using is given recon the susceptibility of Cucumber is realized, for example utilize sacB-neo fusion gene to carry out positive/negative screening, neo(kantlex) resistance is for just screening, and the sucrose toxicity producing due to the expression of sacB gene is for negative screening (Warming et al., Simple and highly efficient BAC recombineering using galK selection.Nucleic acids research, 2005.33 (4): p.e36).Bidirectional screening also can be realized by a bidirectional screening marker gene, for example tolC gene not only can be used for just screening but also can be used for 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 ratio of negative sieve is extremely low even usually failure often.This is mainly production and accumulation due to the lethal process need toxic substance of negative screening, and in cumulative process, cell spontaneous mutation easily occurs produces the resistance to toxic substance, and cell can negatively be survived sieve gene in the situation that possessing.Moreover, the conversion process before negative sieve is also usually introduced the failure that the negative sieve insensitive foreign cell of material is caused to negative sieve process.
Therefore, this area needs a kind of method that can carry out fast and efficiently gene editing.
Summary of the invention
For the above-mentioned and other defect of traditional gene editing method, the invention provides a kind of gene editing method, comprise the following steps:
A., target cell is provided, and described target cell comprises the target nucleic acid with site to be finished;
B. transform described target cell with the nucleic acid fragment of the upstream and downstream homologous sequence that comprises described site to be finished, goal gene, parallel tumor-necrosis factor glycoproteins and bidirectional screening marker gene, so that described nucleic acid fragment and target nucleic acid generation homologous recombination, thereby produce needed gene editing in described site to be finished;
C. just screening to filter out the recon cell of having integrated goal gene and bidirectional screening marker gene on target nucleic acid;
D. allow parallel tumor-necrosis factor glycoproteins and the upstream in site to be finished or the corresponding parallel tumor-necrosis factor glycoproteins generation homologous recombination in downstream on described nucleic acid fragment, with delete flag gene;
E. bear screening and deleted the recon cell of bidirectional screening marker gene with enrichment.
In a kind of preferred implementation, the parallel tumor-necrosis factor glycoproteins in above-mentioned steps b amplifying nucleic acid fragment is the original series in upstream, site to be finished or downstream on target nucleic acid.In another preferred embodiment, on the parallel tumor-necrosis factor glycoproteins in step b amplifying nucleic acid fragment and target nucleic acid, be close to the sequence homology in upstream or the downstream of decorating site, to realize seamless editor.
In another embodiment, the recon cell that in steps d, culturing step c filters out in not containing the substratum of positive-negative selection material is to allow parallel tumor-necrosis factor glycoproteins and the upstream in site to be finished or the corresponding parallel tumor-necrosis factor glycoproteins generation homologous recombination in downstream on described nucleic acid fragment, thus delete flag gene.
In another embodiment, the bidirectional screening marker gene in step b comprises the efficiency that delete to improve bidirectional screening marker gene in one or more Chi site.In preferred implementation, can not affect under the prerequisite of bidirectional screening marker gene function, Chi site is being incorporated into any one or more sites of bidirectional screening marker gene.Described Chi site can be forward or reverse, between multiple Chi site, can be continuous or discontinuous.In dissimilar genome, can use different Chi sites, and in the genome of same type, can use the Chi site sequence of varying strength.
In a kind of embodiment, the inventive method is for the editor of prokaryotic cell prokaryocyte or gene of eucaryote cell group optional position gene, and target nucleic acid is prokaryotic cell prokaryocyte or eukaryotic genome.In another embodiment, the inventive method for carrying out gene editing on plasmid or artificial chromosome (BAC), and target nucleic acid is plasmid or artificial chromosome.
Accompanying drawing explanation
Accompanying drawing 1: three kinds of major ways of gene editing.
Accompanying drawing 2: parallel tumor-necrosis factor glycoproteins auxiliary gene editor's principle schematic.
Accompanying drawing 3: the bidirectional screening marker gene schematic diagram that contains Chi site.
Accompanying drawing 4: parallel tumor-necrosis factor glycoproteins and Chi site auxiliary gene editor's principle schematic.
Accompanying drawing 5: the schematic diagram that carries out gene editing by the inventive method in intestinal bacteria.
Accompanying drawing 6: plasmid pMD18-T collection of illustrative plates (purchased from TaKaRa company).
Accompanying drawing 7: plasmid pEASY-cat-sacB collection of illustrative plates.
Accompanying drawing 8: plasmid pKD46 collection of illustrative plates.
The sequence of accompanying drawing 9:FADE I, shows respectively homologous region, FADE upstream, cat-sacB, homologous region, FADE upstream and homologous region, FADE downstream.
The sequence of accompanying drawing 10:FAA-URA3, shows respectively faa1 upstream from start codon homologous region, faa1 upstream from start codon homologous region, homologous region, faa1 terminator codon downstream.
The sequence of accompanying drawing 11:pMD-ldhAI-cat-sacB-FAR, shows respectively insertion point upstream sequence, cat-sacB, homologous region, insertion point upstream, FAR, homologous region, insertion point downstream.
The sequence of accompanying drawing 12:pMD-tesC-cat-sacB-FAR, shows respectively homologous region, tesC upstream, cat-sacB, homologous region, tesC upstream, FAR, homologous region, tesC downstream.
Embodiment
The present invention utilizes homologous recombination after genome high frequency spontaneous disruption to restart the principle of replication fork, by the method at marker gene two ends series parallel tumor-necrosis factor glycoproteins, realized a step and transformed the recombinated process of gene editing of two steps.With respect to traditional gene editing method (needing twice conversion), just can realize the transformation of expection and the deletion of bidirectional screening marker gene because method provided by the invention only need to once transform, therefore simplify to a great extent the operation steps of gene editing.In addition, method provided by the invention has improved the deletion efficiency of bidirectional screening marker gene.Be not bound by any theory restrictions, the raising of bidirectional screening marker gene deletion efficiency may be to be by intermolecular homologous recombination owing to deleting traditionally the method for bidirectional screening marker gene, thisly be binned in the impact that is subject to a great extent transformation efficiency (being generally no more than 1/1000), and method of the present invention has been recombinated by molecule, each cell contains the fragment that homologous recombination occurs.In addition, the inventive method has improved the efficiency of negative screening by the mode of introducing Chi site in bidirectional screening marker gene, can reach 50%.Because method of the present invention can be carried out the seamless editor of gene, therefore can realize continuously the operations such as the deletion, replacement, insertion of gene.
In the context of the invention, term " target cell " refers to cell to be transformed, contains target nucleic acid in target cell, uses the nucleic acid fragment designing according to the present invention and build to transform this cell, make nucleic acid fragment and target nucleic acid generation homologous recombination, thereby realize required gene editing.
Term " target nucleic acid " refers to the nucleic acid that need to carry out gene editing, and target nucleic acid can be prokaryotic cell prokaryocyte or eukaryotic genome, can be also plasmid or artificial chromosome (BAC) etc.
In the context of the invention, term " nucleic acid " comprises DNA, cDNA and RNA.
Term " gene editing " refers in site to be finished and changes gene order, comprises gene elmination, insertion and replacement.Gene editing in the context of the invention comprises to be edited gene or any base, by original gene or base on gene or base deletion arbitrarily, insertion or replacement target nucleic acid.
Term " seamless editor " or " seamless gene editing " refer on editor's nucleic acid and do not have unwanted unnecessary base.
Term " site to be finished " or " decorating site " refer to the particular location that needs to carry out gene editing on target nucleic acid.
Term " homology ", for example " homology " in " homologous sequence ", refers to two nucleotide sequence similarity or consistence between nucleic acid.Those skilled in the art know, can hybridize the assessment of carrying out homology (as Haines and Higgins (chief editor) " nucleic acid hybridization " (Nucleic Acid Hybridization) (IRL press by DNA-DNA or DNA-RNA, Oxford, Britain) described in), or carry out the assessment of homology by the sequence identity comparing between two nucleic acid.For the object of the invention, " homology " refers between two nucleic acid at least have 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistence, preferably have 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistence, more preferably have 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistence, most preferably have 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistence.The for example BLAST of software and the ClustalW program that can be used for carrying out sequence alignment, two kinds of programs can find from network.
Term " nucleic acid fragment that the present invention builds " or " nucleic acid fragment of design and structure according to the present invention " refer to the nucleic acid fragment of the upstream and downstream homologous sequence, goal gene (when for gene elmination, not having goal gene), parallel tumor-necrosis factor glycoproteins and the bidirectional screening marker gene that comprise site to be finished.Wherein be positioned at the two ends of this nucleic acid fragment with the sequence of the upstream and downstream sequence homology in site to be finished, for target nucleic acid homologous recombination.
Term " goal gene " refers to the gene for carry out gene editing in site to be finished, for example replace gene original on target nucleic acid or be inserted on target nucleic acid in site to be finished, when for delete gene or base on target nucleic acid time, do not need goal gene, on the nucleic acid fragment building in the present invention, goal gene is not for existing.
Term " parallel tumor-necrosis factor glycoproteins " refers to upstream or the downstream sequence homology in site to be finished on one section of sequence on the nucleic acid fragment building in the present invention and target nucleic acid, nucleic acid fragment 5 ' the end that for example the present invention builds contains the parallel tumor-necrosis factor glycoproteins (being the corresponding parallel tumor-necrosis factor glycoproteins in the downstream in site to be finished) with downstream (i.e. 3 ' end) homology in site to be finished, or the nucleic acid fragment 3 ' end that builds of the present invention contains and the parallel tumor-necrosis factor glycoproteins (being the corresponding parallel tumor-necrosis factor glycoproteins of the upstream in site to be finished) of upstream (5 ' holding) homology in site to be finished.On this section of sequence on the nucleic acid fragment that the present invention builds and target nucleic acid, be all called in the present invention " parallel tumor-necrosis factor glycoproteins " with the sequence of its homology.The variable-length of parallel tumor-necrosis factor glycoproteins, for example, within the scope of 30 base to 1000 bases, those skilled in the art can determine the length of parallel tumor-necrosis factor glycoproteins according to specific needs.Term " bidirectional screening marker gene " or " marker gene " refer to not only can just screen but also can bear the gene screening, and can be one or more genes, for example, and tolC, galK, thyA, neo-sacB, tetA-sacB, cat-sacB etc.
" just screen " and there is the implication that those skilled in the art understand conventionally, generally giving recon by antibiotics resistance marker gene gives in the characteristic of corresponding selection defective type substratum growth and realizing to antibiotic resistance or by auxotroph gene, for example utilize and just screening thing kantlex or paraxin, uridylic defective type substratum etc.
" negative screening " has the implication that those skilled in the art understand conventionally, and its feature is generally under specific qualifications, and the existence of negative screening thing has disadvantageous effect to cell, for example, make necrocytosis or obstruction/cell growth inhibiting.The most frequently used negative selection markers is the sacB of subtilis (Bacillus subtilis).When sacB is introduced into heterologous host for example when intestinal bacteria, it can cause necrocytosis in the situation that Exogenous Sucrose exists.
" Chi site " or " Chi sequence " is one section of short DNA fragmentation in genome, and near the probability that homologous recombination occurs this site increases.In dissimilar organic genome, the sequence in Chi site may be different.
Below with reference to accompanying drawing, the present invention is described further.
Fig. 1 shows three kinds of major ways of gene editing, the i.e. deletion of gene or base, replacement or insertion.
Fig. 2 is parallel tumor-necrosis factor glycoproteins auxiliary gene editor's principle schematic.As shown in FIG., according to the present invention, design and the nucleic acid fragment that builds contain at its two ends and the sequence of the upstream and downstream homology in site to be finished, therefore can there is homologous recombination with target nucleic acid (genome or plasmid) in described fragment after being transformed in cell, described nucleic acid fragment is incorporated on target nucleic acid, and the gene in site to be finished or base are deleted, replaced gene or the base in site to be finished or be inserted into site to be finished.After there is the homologous recombination of the above-mentioned the first step, by just screening enrichment recon, if for example one of bidirectional screening marker gene on described nucleic acid fragment is cat, it is chloramphenicol resistance gene, can be with the Screening of Media that contains paraxin the cell through transforming, thereby there is homologous recombination and had the recon cell of chlorampenicol resistant with enrichment.
After just screening, need to remove marker gene.The inventive method utilizes two homologous recombination between parallel tumor-necrosis factor glycoproteins to reject marker gene.One of described parallel tumor-necrosis factor glycoproteins is positioned at upstream or the downstream in site to be screened, one according to the present invention build nucleic acid fragment on, between two parallel tumor-necrosis factor glycoproteinss, be bidirectional screening marker gene.If the nucleic acid fragment that the present invention builds contains parallel tumor-necrosis factor glycoproteins at 5 ' end, 3 ' of this sequence and site to be finished end upstream sequence homology, if the nucleic acid fragment that the present invention builds contains parallel tumor-necrosis factor glycoproteins at 3 ' end, 5 ' of this sequence and site to be finished end upstream sequence homology, thus when these two parallel tumor-necrosis factor glycoproteinss are in same a part, can there is homologous recombination and by them between marker gene deletion.Between described two parallel tumor-necrosis factor glycoproteinss, at least there is 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistence, preferably there is 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistence, more preferably there is 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistence, most preferably there is 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence similarity or consistence.In a kind of particularly preferred embodiment, the parallel tumor-necrosis factor glycoproteins on nucleic acid fragment is the original series in upstream, site to be finished or downstream on target nucleic acid.The length of parallel tumor-necrosis factor glycoproteins can change as required, and for example, within the scope of 30 to 1000 bases, those skilled in the art can determine the length of parallel tumor-necrosis factor glycoproteins according to specific needs.As shown in Figure 2, after there is the first step homologous recombination, on target nucleic acid, contain this two parallel tumor-necrosis factor glycoproteinss, between two parallel tumor-necrosis factor glycoproteinss across marker gene, then allow homologous recombination in these two parallel tumor-necrosis factor glycoproteins generation molecules, for example, by not containing cultivating recon cell for some time in the substratum of positive-negative selection material, thereby remove marker gene.
The inventive method can be carried out the seamless editor of gene.Still with reference to figure 2, if be close to the sequence homology in upstream or the downstream of decorating site on the parallel tumor-necrosis factor glycoproteins on the nucleic acid fragment that the present invention builds and target nucleic acid, can realize seamless editor." the next-door neighbour upstream of decorating site or the sequence in downstream " refers to that between described upstream or downstream sequence and decorating site be continuous, there is no base interval.
In the inventive method, in bidirectional screening marker gene, introduce one or more Chi site and further improved the efficiency (referring to Fig. 3 and Fig. 4) that bidirectional screening marker gene is deleted, make the most effective of final negative screening reach 50%.Can not affect under the prerequisite of bidirectional screening marker gene function, Chi site is being incorporated into any one or more sites of bidirectional screening marker gene.Described Chi site can be forward or reverse, between multiple Chi site, can be continuous or discontinuous.According to different target cell types, can the different Chi site of choice for use, in same type target cell, Chi site sequence that also can choice for use varying strength.
Fig. 5 is the schematic diagram that carries out gene editing by the inventive method in intestinal bacteria.First be that the nucleic acid fragment that the present invention builds transforms competent escherichia coli cell by target dna fragment, then the intestinal bacteria recon cell that screening contains cat-sacB bidirectional screening marker gene on the flat board that contains paraxin, the recon cell filtering out is not first being cultivated containing on the common LB substratum of positive-negative selection thing, then by the LB culture medium culturing that contains negative screening thing sucrose, with the LB plate screening enrichment positive colony that contains sucrose, last available PCR verifies the positive colony obtaining again.
Fig. 6, Fig. 7 and Fig. 8 are the collection of illustrative plates of the plasmid that uses in the embodiment of the present invention.
The inventive method can be used for the editor of optional position gene on the genome of for example intestinal bacteria of prokaryotic cell prokaryocyte or for example yeast of eukaryotic cell, is also used in and on plasmid or artificial chromosome, carries out gene editing.
By reference to the accompanying drawings the present invention is done to detailed description above.It will be appreciated by those skilled in the art that these are described is in order to explain better and to understand the present invention, is not intended to scope of the present invention to do any restriction.Below in connection with specific embodiment, the present invention is further illustrated.Equally, these embodiment, just in order to make those skilled in the art can understand better the present invention, do not form any restriction to the scope of the invention.Scope of the present invention is limited only by the accompanying claims.
Those skilled in the art approval, can carry out multiple variation and change and not depart from the spirit and scope of the present invention form of the present invention, and these variations and changing owing to belonging to equivalents of the present invention fall in the scope of the claims in the present invention protection.
Except this otherwise noted, in the application, term used has general sense understood by one of ordinary skill in the art.The experimental implementation in molecular biology and genetically engineered field, for example the general technology of plasmid construction, conversion and screening is well known to those skilled in the art, also can be referring to the book of reference of this area for example " molecular cloning experiment guide ", the 3rd edition, J. Pehanorm Brooker.The genome sequence of intestinal bacteria (Escherichia coli) can be
hTTP: //WWW.NCBI.NLM.NIH.GOV/GENOME/167 PROJECT_ID=57779obtain.The genome sequence of ocean bacillus (Marinobacter aquaeolei VT8) can be
hTTP: //WWW.NCBI.NLM.NIH.GOV/GENOME/ TERM=MARINOBACTER+AQ uAEOLEIobtain.
Embodiment
The seamless deletion of embodiment 1 intestinal bacteria (Escherichia coli) fadE
(1) the synthetic homologous region, fadE two ends of containing, the DNA of the parallel tumor-necrosis factor glycoproteins of 5 ' end and cat-sacB, called after fadE I (SEQ ID NO.:1, also referring to Fig. 9).
(2) fragment fadE I transforms the E.coli MG1655 competent cell containing pKD46, coats the LB solid medium containing paraxin after hatching.After growing, obtain recombinant bacterium MG1655 Δ fadE::cat-sacB through PCR and sequence verification.
(3) purifying MG1655 Δ fadE::cat-sacB, negative screening process, gets single colony inoculation in 100mL LB liquid nutrient medium as shown in Figure 5, cultivates 12h; Get 1mL nutrient solution and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h; Get 300 μ L nutrient solutions and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h.
(4) get the LB solid medium of suitable dilution spread 10% sucrose of above-mentioned nutrient solution, wait to grow single bacterium colony, with fadEp1(TTGAAACCGAAATCATTACCGACGC, SEQ ID NO.:2) and fadEp2(CGTGTTATCGCCAGGCTTTAGGAGG, SEQ ID NO.:3) carry out PCR detection for primer PCR, PCR response procedures is: 94 ℃ of 4min94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 2min35cycles, the following all embodiment of 72 ℃ of 5min(PCR program used is all with identical herein) sequence verification obtains seamless deletion bacterial strain.
The seamless deletion of embodiment 2 intestinal bacteria tesC
(1) utilize PCR to react take E.coli MG1655 genome as template, use upstream and downstream primer tesCp1(GCACTGCTCATTACCCTGTCCCTG, SEQ ID NO.:4) and tesCp2(TGGATGTCACCCTGCTCAACGAG, SEQ ID NO.:5) amplification intestinal bacteria tesC gene and upstream and downstream partial sequence acquisition tesC I fragment, this fragment and pMD18-T are obtained to pMD18-tesC.
(2) utilize PCR to react take pMD18-tesC as template, use upstream and downstream primer tesCp3(AAAATTGCCACTATGCAAATTAATTACAGGG, SEQ ID NO.:6) and tesCp4(ACGTTTTGTGGTGCCGGATGCTC, SEQ ID NO.:7) oppositely increasing obtains tesC II fragment.
(3) utilize PCR to react take pEASY-cat-sacB as template, use phosphorylation upstream and downstream primer P5(GTGACGGAAGATCACTTCGCAGA, SEQ ID NO.:8) and tesCp6(
aAAATTGCCACTATGCAAATTAATTACAGGGTTAATACCGCCAGATTACGaTCAAAGGGAAAACTGTCCATATGC, SEQ ID NO.:9, underscore part is tesC upstream region of gene homologous sequence) amplification acquisition tesC III fragment.
(4) junction fragment tesC II and tesC III, Transformed E .coli DH5 α bacterial strain, obtains pMD-tesC-cat-sacB plasmid.
(5) utilize PCR to react take pMD-tesC-cat-sacB as template, use upstream and downstream primer tesCp1 and tesCp2 amplification to obtain tesC IV fragment.
(6) fragment tesC IV electricity transforms the E.coli MG1655 competent cell containing pKD46, hatches rear coating and the LB solid medium that contains paraxin.After growing, PCR detects, and sequence verification obtains recombinant bacterium MG Δ tesC::cat-sacB.
(7) purifying MG Δ tesC::cat-sacB, negative screening process, gets single colony inoculation in 100mL LB liquid nutrient medium as shown in Figure 5, cultivates 12h; Get 1mL nutrient solution and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h: get 300 μ L nutrient solutions and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h.
(8) get the LB solid medium of suitable dilution spread 10% sucrose of above-mentioned nutrient solution, wait to grow single bacterium colony, detect take tesCp1 and tesCp2 as primer PCR, sequence verification obtains seamless deletion bacterial strain.
Embodiment 3 ocean bacillus (Marinobacter aquaeolei VT8) the seamless replacement of FAR gene intestinal bacteria tesC
(1) use primers F ARF(ATGGCAATACAGCAGGTACATCACG, SEQ ID NO.:10) and FARR(TCAGGCAGCTTTTTTGCGCTG, SEQ ID NO.:11), obtain FARS I fragment take ocean bacillus gene group as template.
(2), take pMD-tesC-cat-sacB as template, obtain FARS II fragment take tesCp4 and tesCp6 as primer amplification.
(3) junction fragment FARS I and FARS II, Transformed E .coli DH5 α bacterial strain, obtains pMD-tesC-cat-sacB-FAR plasmid (SEQ ID NO.:27, also referring to accompanying drawing 12).
(4) utilize PCR to react take pMD-tesC-cat-sacB-FAR as template, use upstream and downstream primer tesCp1 and tesCp2 amplification to obtain FARS III fragment.
(5) fragment FARS III electricity transforms the E.coli MG1655 competent cell containing pKD46, hatches rear coating and the LB solid medium that contains paraxin.After growing, PCR detects, and sequence verification obtains recombinant bacterium MG Δ tesC::cat-sacB-FAR.
(6) purifying MG Δ tesC::cat-sacB-FAR, negative screening process, gets single colony inoculation in 100mL LB liquid nutrient medium as shown in Figure 5, cultivates 12h; Get 1mL nutrient solution and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h: get 300 μ L nutrient solutions and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h.
(7) get the LB solid medium of suitable dilution spread 10% sucrose of above-mentioned nutrient solution, wait to grow single bacterium colony, detect take tesCp1 and tesCp2 as primer PCR, sequence verification obtains seamless replacement bacterial strain.
Embodiment 4 ocean bacillus FAR gene seamless insertion intestinal bacteria ldhA promotor downstreams
(1) utilize PCR to react take E.coli MG1655 genome as template, use upstream and downstream primers F ARIp1 (GCCGAATATCATGGTGGAAAATGG, SEQ ID NO.:12) and FARIp2 (CTGGCGATTGCTCCGTCTGC, SEQ ID NO.:13) amplification intestinal bacteria ldhA promotor upstream and downstream partial sequence acquisition KldhAI fragment, this fragment and pMD18-T are obtained to pMD18-KldhAI.(2) utilize PCR to react take pMD18-KldhAI as template, use upstream and downstream primers F ARIp3 (CATATGAATATCCTCCTTAGTTCCTATTCC, SEQ ID NO.:14) and oppositely amplification acquisition FARI II fragment of FARIp4 (AAGACTTTCTCCAGTGATGTTGAATCACAT, SEQ ID NO.:15).
(3) utilize PCR to react take pMD-cat-sacB as template, use phosphorylation upstream and downstream primer P5 and FARIp6 (
cATATGAATATCCTCCTTAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAaTCAAAGGGAAAACTGTCCATATGC, SEQ ID NO.:16, underscore part is insertion point upstream homologous sequence) amplification acquisition FARI III fragment
(4) junction fragment FARI II and FARI III, Transformed E .coli DH5 α bacterial strain, obtains pMD-ldhAI-cat-sacB plasmid.
(5) utilize PCR to react take pMD-ldhAI-cat-sacB as template, use upstream and downstream primers F ARIp3 and p5 amplification to obtain FARI IV fragment.
(6) use phosphorylation primers F ARF and FARR, obtain FARI V fragment (7) junction fragment FARI IV and FARI V take ocean bacillus gene group as template, Transformed E .coli DH5 α bacterial strain, obtain pMD-ldhAI-cat-sacB-FAR(SEQ ID NO.:28, also referring to accompanying drawing 11) plasmid.
(8) utilize PCR to react take pMD-ldhAI-cat-chi-sacB-FAR as template, FARIp1 and FARIp2 amplification obtain FARI VI fragment.
(9) fragment FARS VI electricity transforms the E.coli MG1655 competent cell containing pKD46, hatches rear coating and the LB solid medium that contains paraxin.After growing, PCR detects, and sequence verification obtains FAR and inserts bacterial strain MGKLFARI.
(10) purifying MGKLFARI, negative screening process, gets single colony inoculation in 100mL LB liquid nutrient medium as shown in Figure 5, cultivates 12h; Get 1mL nutrient solution and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h: get 300 μ L nutrient solutions and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h.
(11) get the LB solid medium of suitable dilution spread 10% sucrose of above-mentioned nutrient solution, wait to grow single bacterium colony, detect take FARIp1 and FARIp2 as primer PCR, sequence verification obtains seamless insertion bacterial strain.
The structure of embodiment 5pMD-cat-chi-sacB
(1) utilize PCR reaction to use upstream and downstream primer pccs-p1(GTGACGGAAGATCACTTCGCAGA, SEQ ID NO.:17 take pEASY-cat-sacB as template) and pccs-p2(
cCACCAGCcAGTAACAAACCCGCGCGATTT, SEQ ID NO.:18, underscore is chi site sequence) and obtain catchi fragment, this fragment and pMD18-T are obtained to pMD18-cat-chi.
(2) utilize PCR reaction to use upstream and downstream primer pccs-P3(take pMD18-cat-chi as template
cCACCAGCcAGTAACAAACCCG, SEQ ID NO.:19, underscore is chi site sequence) and pccs-P4(ATCTCTAGAGGATCCCCGGGTACC, SEQ ID NO.:20) acquisition pMD18-catchiF fragment.
(3) utilize PCR reaction to use the upstream and downstream primer P5(GTGACGGAAGATCACTTCGCAGA of phosphorylation take pEASY-cat-sacB as template) and pccs-p6(ATCAAAGGGAAAACTGTCCATATGC, SEQ ID NO.:21) acquisition chisacB fragment.
(4) connect chisacB fragment and pMD18-catchiF fragment, Transformed E .coli DH5 α bacterial strain, obtains pMD-cat-chi-sacB plasmid.
Embodiment 6 height are followed seamless deletion intestinal bacteria fadE
(1) the synthetic homologous region, fadE two ends of containing, the single stranded DNA of the parallel tumor-necrosis factor glycoproteins of 5 ' end and cat-chi-sacB, called after fadE II(SEQ ID NO.:22).
(2) fragment fadE II electricity transforms the E.coli MG1655 competent cell containing pKD46, hatches rear coating and the LB solid medium that contains paraxin.After growing, PCR detects, and sequence verification obtains recombinant bacterium MG Δ fadE::cat-chi-sacB
(3) purifying MG Δ fadE::cat-chi-sacB, negative screening process, gets single colony inoculation in 100mL LB liquid nutrient medium as shown in Figure 5, cultivates 12h; Get 1mL nutrient solution and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h: get 300 μ L nutrient solutions and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h.
(4) get the LB solid medium of suitable dilution spread 10% sucrose of above-mentioned nutrient solution, wait to grow single bacterium colony, detect take fadEp1 and fadEp2 as primer PCR, sequence verification obtains seamless deletion bacterial strain.
Embodiment 7 height are followed seamless deletion intestinal bacteria tesC
(1) utilize PCR to react take E.coli MG1655 genome as template, use upstream and downstream primer tesCp1 and tesCp2 amplification intestinal bacteria tesC gene and upstream and downstream partial sequence to obtain tesC I fragment, this fragment and pMD18-T are obtained to pMD18-tesC.
(2) utilize PCR to react take pMD18-tesC as template, use upstream and downstream primer tesCp3 and tesCp4 oppositely to increase and obtain tesC II fragment.
(3) utilize PCR to react take pMD-cat-chi-sacB as template, use phosphorylation upstream and downstream primer P5 and tesCp6 amplification to obtain tesC III fragment.
(4) junction fragment tesC II and tesC III, Transformed E .coli DH5 α bacterial strain, obtains pMD-tesC-cat-chi-sacB plasmid.
(5) utilize PCR to react take pMD-tesC-cat-chi-sacB as template, use upstream and downstream primer tesCp1 and tesCp2 amplification to obtain tesC IV fragment.
(6) fragment tesC IV electricity transforms the E.coli MG1655 competent cell containing pKD46, hatches rear coating and the LB solid medium that contains paraxin.After growing, PCR detects, and sequence verification obtains recombinant bacterium MG Δ tesC::cat-chi-sacB.
(7) purifying MG Δ tesC::cat-chi-sacB, negative screening process, gets single colony inoculation in 100mL LB liquid nutrient medium as shown in Figure 5, cultivates 12h; Get 1mL nutrient solution and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h: get 300 μ L nutrient solutions and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h.
(8) get the LB solid medium of suitable dilution spread 10% sucrose of above-mentioned nutrient solution, wait to grow single bacterium colony, detect take tesCp1 and tesCp2 as primer PCR, sequence verification obtains seamless deletion bacterial strain.
Embodiment 8 height are followed the seamless replacement of ocean bacillus FAR gene intestinal bacteria tesC
(1) use primers F ARF and FARR, obtain FARS I fragment take ocean bacillus gene group as template.
(2) take pMD-tesC-cat-chi-sacB as template, with primer tesCp4 and sacBR(ATCAAAGGGAAAACTGTCCATATGCAC, SEQ ID NO.:23) amplification acquisition FARS II fragment.
(3) junction fragment FARS I and FARSII, Transformed E .coli DH5 α bacterial strain, obtains pMD-tesC-cat-chi-sacB-FAR plasmid.
(4) utilize PCR to react take pMD-tesC-cat-chi-sacB-FAR as template, use upstream and downstream primer tesCp1 and tesCp2 amplification to obtain FARS III fragment.
(5) fragment FARS III electricity transforms the E.coli MG1655 competent cell containing pKD46, hatches rear coating and the LB solid medium that contains paraxin.After growing, PCR detects, and sequence verification obtains recombinant bacterium MG Δ tesC::cat-chi-sacB-FAR.
(6) purifying MG Δ tesC::cat-chi-sacB-FAR, negative screening process, gets single colony inoculation in 100mL LB liquid nutrient medium as shown in Figure 5, cultivates 12h; Get 1mL nutrient solution and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h: get 300 μ L nutrient solutions and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h.
(7) get the LB solid medium of suitable dilution spread 10% sucrose of above-mentioned nutrient solution, wait to grow single bacterium colony, detect take tesCp1 and tesCp2 as primer PCR, sequence verification obtains seamless replacement bacterial strain.
Embodiment 9 height are followed ocean bacillus FAR gene seamless insertion intestinal bacteria ldhA promotor downstream
(1) utilize PCR to react take E.coli MG1655 genome as template, use upstream and downstream primers F ARIp1 and FARIp2 amplification intestinal bacteria ldhA promotor upstream and downstream partial sequence to obtain KldhAI I fragment, this fragment and pMD18-T are obtained to pMD18-KldhAI.
(2) utilize PCR to react take pMD18-KldhAI as template, use upstream and downstream primers F ARIp3 and FARIp4 oppositely to increase and obtain FARI II fragment.
(3) utilize PCR to react take pMD-cat-chi-sacB as template, use phosphorylation upstream and downstream primer P5 and FARIp6 amplification to obtain FARI III fragment
(4) junction fragment FARI II and FARI III, Transformed E .coli DH5 α bacterial strain, obtains pMD-ldhAI-cat-chi-sacB plasmid.
(5) utilize PCR to react take pMD-ldhAI-cat-chi-sacB as template, use upstream and downstream primers F ARIp3 and p5 amplification to obtain FARI IV fragment.
(6) use phosphorylation primers F ARF and FARR, obtain FARI V fragment take ocean bacillus gene group as template
(7) junction fragment FARI IV and FARI V, Transformed E .coli DH5 α bacterial strain, obtains pMD-ldhAI-cat-chi-sacB-FAR plasmid.
(8) utilize PCR to react take pMD-ldhAI-cat-chi-sacB-FAR as template, FARIp1 and FARIp2 amplification obtain FARI VI fragment.
(9) fragment FARS VI electricity transforms the E.coli MG1655 competent cell containing pKD46, hatches rear coating and the LB solid medium that contains paraxin.After growing, PCR detects, and sequence verification obtains FAR and inserts bacterial strain MGKLFARI.
(10) purifying MGKLFARI, negative screening process, gets single colony inoculation in 100mL LB liquid nutrient medium as shown in Figure 5, cultivates 12h; Get 1mL nutrient solution and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h: get 300 μ L nutrient solutions and be inoculated in the LB liquid nutrient medium containing 10% sucrose, cultivate 12h.
(11) get the LB solid medium of suitable dilution spread 10% sucrose of above-mentioned nutrient solution, wait to grow single bacterium colony, detect take tFARIp1 and FARIp2 as primer PCR, sequence verification obtains seamless insertion bacterial strain.
The seamless yeast FAA1 that knocks out of embodiment 10
(1) the synthetic homologous region, FAA1 two ends of containing, the single stranded DNA of the parallel tumor-necrosis factor glycoproteins of 5 ' end and URA3, called after FAA-URA3(SEQ ID NO.:24, also referring to accompanying drawing 10).
(2) fragment FAA-URA3 electricity transformed saccharomyces cerevisiae competent cell, coats SD substratum after hatching.After growing, PCR detects, and sequence verification obtains FAA1 and knocks out bacterium TAMkf.
(3) purifying TAMkf, gets single colony inoculation in SD+ uridylic liquid nutrient medium, cultivates 24h; Getting 1mL nutrient solution, to be inoculated in SD+ uridylic+5-FOA(be 5-fluororotic acid) liquid nutrient medium, cultivate 24h: get 300 μ L nutrient solutions and be inoculated in SD+ uridylic+5-FOA liquid nutrient medium, cultivate 24h.
(4) get above-mentioned nutrient solution and line SD+ uridylic+5-FOA solid medium, cultivate 50h, point-to-point SD+ uridylic and the SD solid medium of being incubated at after picking list bacterium colony.
(5) being chosen at SD solid medium does not grow, at the bacterium colony of SD+ uridylic substratum growth, with FAAp1 (TTAGGATACAATAAAAACTAGAACAAACAC, SEQ ID NO.:25) and FAAp2 (CTATCATGGAAATGTTGATCC, SEQ ID NO.:26) be primer PCR, sequence verification obtains seamless deletion bacterial strain.
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| CN114107253A (en) * | 2021-12-17 | 2022-03-01 | 复旦大学附属华山医院 | A system and method for gene editing using engineered cells |
| US20230193256A1 (en) * | 2021-12-17 | 2023-06-22 | Jianhong Zhu | System and method for gene editing by using engineered cell |
| CN114107253B (en) * | 2021-12-17 | 2024-03-15 | 复旦大学附属华山医院 | System and method for gene editing by using engineering cells |
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| WO2015158115A1 (en) | 2015-10-22 |
| CN103898140B (en) | 2017-03-22 |
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