CN101892221A - Traceless modification method of chromosome - Google Patents
Traceless modification method of chromosome Download PDFInfo
- Publication number
- CN101892221A CN101892221A CN2010102132878A CN201010213287A CN101892221A CN 101892221 A CN101892221 A CN 101892221A CN 2010102132878 A CN2010102132878 A CN 2010102132878A CN 201010213287 A CN201010213287 A CN 201010213287A CN 101892221 A CN101892221 A CN 101892221A
- Authority
- CN
- China
- Prior art keywords
- chromosomal
- traceless
- gene
- scei
- homologous
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention provides a traceless modification method of a chromosome, comprising the following steps: firstly building a recombinant box composed of a positive selection marker, a counterselection marker, an endonuclease I-SceI, an induced promoter, an I-SceI recognition site and a target nucleic acid displacement sequence which is homologous with a target chromosome nucleic acid sequence; inputting DNA fragments into a host containing recombinant plasmids by electrotransformation; knocking out the following gene sequences from an expected recombinant through a double-strand break repair phenomenon mediated by I-SceI: the positive selection marker, the induced promoter, an I-SceI gene and the counterselection marker; and finally screening the final recombinant from a sucrose medium. The method can help insert, knock out or replace the target gene of the chromosome without any selection markers or exogenous DNA sequences remained, thus being an effective method for traceless modification of the DNA sequence of a host cell.
Description
Technical field
The present invention relates to adopt the gene recombination means that one or more target genes on the karyomit(e) are inserted, knock out or method of replacement, especially a kind of traceless modification method that can not stay any selection markers or exogenous DNA array to original chromosome belongs to chromogene group renovation technique field.
Background technology
Chromogene group renovation technique, for example: technology such as gene interference, foreign gene insertion, transgenation, extremely important for bioenergy, metabolic engineering and Agricultural engineering field.Red/ET recombined engineering (Recombineering) is a kind of novel gene engineering of setting up in recent years, it is the interior homologous recombination reaction of high efficiency body that mediates by the recombinant protein (Exo, Beta, Gam) that phage produces, the target DNA sequence is carried out operations such as gene inserts, knocks out, replacement, do not need to select suitable restriction site.
One of bottleneck in the gene recombination engineering of eukaryotic cell and some bacteriums is to have high-frequency non-homogeneous reorganization and inefficient homologous recombination, thereby need to screen in a large number so that isolate required mutator gene, this method is inoperative in the bacterium of slowly growth such as mycobacteria, and is much lower because frequency is compared in homologous recombination and non-homogeneous reorganization.
For fear of large vol ground screening recombination, generally need in transfer box, comprise selection markers and be beneficial to confirm knocking in or knocking out of target spot gene regions.Yet the selection markers of insertion can influence genomic further transformation.In order to prevent that karyomit(e) behind the genome manipulation from (for example: antibiotics resistance gene) or exogenous DNA array (for example FRT or loxP site) having remaining selection markers, method commonly used at present is to utilize the locus specificity of Flp recombinase target spot (FRP) or Cre recombinase target spot (loxP) mediation, thereby reaches the purpose of accurate shearing selection markers.Yet above method has characteristics and defective separately.One still exists at least one FRT site or loxP site, promptly so-called remaining trace after selection markers is sheared; Its two, even under the non-existent situation of recombinase, contain a plurality of remaining traces karyomit(e) since the reorganization phenomenon between the vestige point cause the random rearrangement of chromogene group easily or knock out.Therefore, press for and a kind ofly can transform target gene or genome sequence and do not stay the method for remaining traces such as selection markers or exogenous DNA array.
To this, a kind of method at present commonly used is to set up an anti-selection markers in the box passing to change, and like this, those anti-screening and cloning of having lost exogenous array are identified owing to producing the dominant lethal effect.For example, at streptomycetes, the glkA gene can be used as anti-selection markers gene.The substratum that contains glucalogue 2-deoxyglucose can cause the transmission box in the bacterial strain to be lost, and this has lethal effect to the glkA+ bacterial strain.Similarly, can use ura 3 genes to carry out the positive and negative screening at Saccharomyces cerevisiae.In the substratum that uridylic lacks, screen wild-type, in comprising 5-fluororotic acid (5-FOA) substratum, screen auxotroph, because 5-FOA is the analogue of uridylic precursor and can be converted into Fluracil (a kind of potent inhibitor of zymic) that wild-type can not be grown at 5-fluororotic acid substratum.
There is obvious defects in aforesaid method, and they involve exchange process twice, does not have positive-selecting in last reconstitution steps, and is confined to work in glkA or the pyrF null mutation bacterial strain.In addition, each knocks out step and needs the multiple plasmid to transform and knock out, and is consuming time and labour intensity is big.Therefore, need a kind of new genome conformity (or knocking out) method, require all steps to have the selection performance, especially in final regrouping process; And, unique change of host genome be insert or knock out ideal basis because of, can not stay any selection markers or remaining trace).
Summary of the invention
Purpose of the present invention is exactly in order to solve the above-mentioned problems in the prior art, and a kind of chromosomal traceless modification method is provided.
Technical solution of the present invention is: chromosomal traceless modification method, at first, make up comprise positive-selecting mark, anti-selection markers, endonuclease I-SceI, inducible promoter, I-SceI recognition site, with the reorganization box of target chromosome nucleotide sequence homologous target nucleic acid constant series; Then, the method for electricity consumption conversion comprises above-mentioned dna fragmentation input the host of recombinant plasmid; Then, repair phenomenon by the double-strand break of I-SceI mediation and from desirable recombinant chou, knock out following gene order: positive-selecting mark, inducible promoters, I-Scel gene, anti-selection markers; At last, the final recombinant chou of screening on sucrose medium.That described reorganization box transforms is Escherichia coli.
Further, above-mentioned chromosomal traceless modification method, modification to target chromosome comprises: one or many ground " inserts one or more Nucleotide to the target cell genome " or " knocking out one or more Nucleotide from the target cell genome " or " one or more Nucleotide in the displacement target cell genome ", and described target cell genome means nucleotide sequence or the non-genomic nucleotide sequence between native gene, native gene.
Further, above-mentioned chromosomal traceless modification method, the specific chromosomal region of modified microorganism according to the following steps:
1) prepares a linear DNA fragment, comprise and the λ-relevant homology arm B and C that can import targeted microorganisms of red reorganization, positive-selecting mark, anti-selection markers, the I-SceI gene, the I-SceI recognition site knocks out the relevant inducible promoters of selection markers, homologous fragment A with homologous recombination;
2) import linear DNA fragment to the microorganism that contains the described reorganization box of claim 1, by λ-Red reorganization replace the dna fragmentation homology arm and and homology arm homologous karyomit(e) between specific site;
3) in the conditioned medium of inducing I-SceI endonuclease expression of enzymes, cultivate specific chromosomal foci metathetical microorganism, cause double-strand break, homologous fragment A and and Segment A homologous chromosomal region between homologous recombination be used to knock out screening property mark, wherein homology arm B and the chromosomal 50-500bp homology that knocks out target spot one end, the 50-500bp homology of the homology arm C and the other end, homologous fragment A is the zone of an end 300-500bp of connection and homology arm B and homology arm C homologous chromosomal region.Wherein,
Described selection markers comprises positive-selecting mark and negative selection markers, the positive-selecting mark is to screen from comprise visual certification mark, allow recombinant chou in the substratum that lacks nutrition source, to grow, give recombinant chou certain compound produced resistance, negative selection markers be a kind of in the presence of substrate pair cell the mark of lethal effect is arranged;
Described screening substrate comprises minimum medium, lacks one or more target cell or one or more essential nutrition sources of microbiotic; And,
Described step 1) and step 2) can repeat repeatedly, prepare a plurality of different linear DNA fragments, with a series of specific chromosomal region of modified microorganism.
The present invention adopts no vestige gene recombination means that one or more target genes on the karyomit(e) are modified; comprise one or many ground " insertion ", " knocking out " or " displacement "; antibiotics resistance gene) or exogenous DNA array (for example FRT or loxP site) (for example:, be the effective ways of seamless modification host cell DNA sequence can not stay any selection markers after modifying.
According to the present invention, but at first in target cell, transform the recombination system of an abduction delivering, yet the foreign DNA that will contain positive-selecting mark and anti-selection markers is introduced target cell.In target cell, cause a series of processes as inducing recombination system; Screen the reorganization target cell of being positive; Induce and express a special endonuclease (for example I-Scel) and an anti-selection markers (for example sacB).Then,, reject the positive and anti-selection markers in the DNA target sequence by in target cell, causing a fragmentation of double-stranded (DSB) phenomenon, last, by being inoculated into sucrose medium, the recombinant bacterial strain that screening is expected.
The modification of target gene can divide three steps.The first step makes up a linear DNA sequence box that comprises following factor by PCR: positive-selecting mark (CM), inducible promoter (Pi), I-Scel gene (SceI), anti-selection markers (SacB), I-Scel recognition site (I), gene and three homology arms (representing with A, B and C respectively) in order to modify.In second step, the method that electricity consumption transforms comprises above-mentioned dna fragmentation input the host of recombinant plasmid.Because recombinant plasmid (for example pKD46) contains the coding lambda particles phage Red required gene of recombinating, thus replace target gene by the homologous recombination phnomena of Red enzyme mediation by above-mentioned dna fragmentation, and filter out by the metathetical cell containing on the paraxin substratum.The 3rd step, repair phenomenon by the double-strand break of I-SceI mediation and from desirable recombinant chou, knock out following gene order: positive-selecting mark (CM), inducible promoters (Pi), I-Scel gene (SceI), anti-selection markers (SacB), the final recombinant chou of screening on sucrose medium at last.This flow process can repeat, so that make up the mutant strain that contains a plurality of modification points.After having finished all modifications, remaining Red plasmid (for example pKD46) can be removed from the mutant of growth in 37 ℃ of following suitable medium in the host.
Description of drawings
Fig. 1 is the present invention carries out traceless modification to karyomit(e) a step synoptic diagram.Wherein,
Step (A) is assembled the linear DNA sequence box that comprises following factor: positive-selecting mark (CM), inducible promoters (Pi), I-SceI gene (Scel), anti-selection markers (SacB), I-SceI recognition site (I), replacement gene fragment, three homology arms (with A, B and C representative) by PCR.The PCR primer uses underscore, tilted letter (P-f1, P-r1, P-f2 and P-r2) and arrow mark;
Step (B), the genomic targets on the E.coli karyomit(e) is replaced by above-mentioned linear DNA sequence box, and screens recombinant chou containing on the LB substratum of paraxin;
Step (C), repair phenomenon by the double-strand break of I-SceI mediation and from desirable recombinant chou, knock out following gene order: positive-selecting mark (CM), inducible promoters (Pi), I-Scel gene (SceI), anti-selection markers (SacB), the final recombinant chou of screening on sucrose medium at last.
Embodiment
The present invention carries out seamless modification by using positive-selecting and anti-selection markers to host chromosome.At first, with the dna fragmentation displacement target gene that contains positive resistance marker, detect recombinant chou at screening property substratum, do not contain the final recombinant chou of any selection markers or exogenous DNA array then by I-SceI inductive double-strand break and anti-selection markers gene sacB screening.
The present invention has reduced some unnecessary steps such as plasmid and has transformed and repeated screening according to intravital DNA reorganization of target cell and Selecting Mechanism and Procedure.
The present invention has used anti-screening marker gene, and for example the sacB gene of Bacillus subtilis makes a large amount of bacteriums produce susceptibility to sucrose.SacB genes encoding levansucrase, catalysis sucrose hydrolysis or polymerization form lethality product Polylevulosan.E.coli and numerous Gram-negative bacteria can't be grown when sacB genetic expression, therefore can directly screen the cell of losing the sacB gene by homologous recombination.
The present invention has used the endonuclease I-SceI that goes back to the nest of intron coding, a fragmentation of double-stranded (DSB) can be transformed into genome.Fragmentation of double-stranded is the substrate of microorganism recombination system, can be by homologous recombination reparation and broken ends both wings homologous sequence area.Under the repair system of host's fragmentation of double-stranded mediation helped, unmarked modification can be incorporated into the genome of Gram-negative bacteria, for example Escherichia coli and Salmonella typhimurium.
The invention provides the target cell that contains the inducibility recombination system.The nucleotide sequence of first external source, comprise positive-selecting mark, anti-selection markers, special endonuclease (I-SceI), inducible promoters, I-SceI recognition site and with target chromosome nucleotide sequence homologous target nucleic acid constant series, import to target cell, express derivable recombination system, the positive reorganization of screening target cell is expressed special endonuclease (for example I-SceI) and anti-selection markers (for example sacB).Knock out positive and anti-selection markers by fragmentation of double-stranded mechanism, screen the mutant of losing the sacB gene on the sucrose medium containing.
The present invention has adopted the visible certification mark, can carry out the positive and negative screening or scanning as fluorescence activity cell screening (FACS) or micro-fluidic technologies.Certification mark comprises various enzymes, prothetic group, fluorescent mark, luminescent marking, bioluminescence marker etc.Fluorescin includes but is not limited to yellow fluorescence protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP) etc.Biological fluorescent labelling includes but is not limited to luciferase, fluorescein, aequorin etc.The enzyme system of visual detection signal is including, but not limited to tilactase, glucocorticosteroid enzyme, Phosphoric acid esterase, peroxidase, Pseudocholinesterase etc.
Among the present invention, the positive-selecting mark is the gene that can give certain compound of cell resistance, does not exist under the situation at this gene, and the compound pair cell has lethal effect.For example, the cell of expressing antibiotics resistance gene can be survived in the presence of microbiotic, and the cell that lacks this gene can not be survived.For example chloramphenicol resistance gene can carry out positive-selecting in the presence of paraxin.Antibiotics resistance gene includes but is not limited to ampicillin resistance gene, neomycin resistance gene, blasticidin resistant gene, hygromycin gene, puromycin resistance gene, tetracycline resistance gene etc.
Among the present invention, anti-selection markers is the gene that target cell is had lethal effect in the presence of specific substrates.For example, the sacB gene has lethal effect in the presence of sucrose.Therefore do not express the SacB gene containing the cell of growing on the sucrose medium.Anti-selection markers is including, but not limited to glkA, pyrF, thyA, sacB, mazF, rpsL etc.
Among the present invention, special endonuclease is by the gene of intron coding, induces double-strand break in the accurate position of homologous chromosomes.Special endonuclease is including, but not limited to I-Sce I, I-Ceu I, P I-Psp I, P I-Sce I, I-Ppo I etc.
Among the present invention, inducible promoter is a DNA element of having ready conditions and expressing that carries out target sequence by the interpolation inductor.Inducible promoter has but is not limited in the microorganism: rha, tet, lac, ara, Gal1, CUP1, CTR1/3, MET25 etc.
Among the present invention, target cell can be any eucaryon or prokaryotic cell prokaryocyte.For example, target cell can be bacterial cell such as E.coli, insect cell such as Drosophila melanogaster cell, vegetable cell, yeast cell, Amphibians cell such as Xenopus laevis cell, threadworms cell such as Caenorhabditis elegans cell, mammalian cell such as Chinese hamster ovary cell (CHO), African green monkey kidney cell (COS), human fetal cell (293T) or other people's cell.The cell of cultivating with transplanting can use in the present invention.
The present invention can continuously change the one or more target nucleic acid sequences (for example native gene) in the target cell, comprise and add one or more Nucleotide to target cell, from target cell, knock out target nucleotide, change in the target cell nucleotide sequence, non-genomic artificial DNA between one or more native genes, gene.
In order to prove the feasibility of method among the present invention, replace with the relA gene of Yersinia pestis bacterial strain KIM6+ below in conjunction with Figure of description that the target gene group between A and C homologous region is an example in the Escherichia coli K-12 bacterial strain (MG1655), technical solution of the present invention is described further.Wherein, displacement fragment (relA gene) can be produced by pcr amplification, and uses forward (P-f1) and reverse (P-r1) primer.
As shown in Figure 1, the present invention has made up screening box (CM-P by recombinant PCR
RhaB-SceI-sacB-I), wherein comprise five DNA districts: positive-selecting mark (CM), inducible promoter (P
RhaB), I-SceI gene, negative selection markers (sacB) and I-SceI recognition site (I).
At first, by conventional PCR from the pDEST10 plasmid, increase chloramphenicol resistance gene (CM) fragment, amplification contains P from the pRED1 plasmid
RhaBWith the fragment of an I-SceI gene, amplification contains the fragment of sacB gene and I-SceI recognition site (I) from plasmid pSC.
Then, the gene fragment of these amplifications of purifying is assembled the said gene fragment by the recombinant PCR reaction, obtains required screening box (CM-P
RhaB-SceI-sacB-I).The 0.5kb homologous fragment that is positioned at left side, target spot district can obtain from the amplification of E.coli K-12 bacterial strain MG1655 karyomit(e) by conventional PCR.This 0.5kb homologous fragment--comprise the flanking sequence of a 20bp, 0.5kb segmental 3 ' holds and treats that 5 ' end of metathetical dna fragmentation is overlapping, and 0.5kb segmental 5 ' holds with 3 ' end of above-mentioned screening box overlapping.
At last, adopt forward primer (P-f2) and anti-phase primer (P-r2) to be assembled into the displacement box that comprises 3 PCR fragments (homologous fragment, screening box and displacement fragment) by recombinant PCR.
The preparation of electroreception attitude cell.Contain under 30 ℃ of the E.coli MG1655 host cells of Red plasmid pKD46 and in the LB substratum that adds penbritin, grow, be in the cell of logarithmic phase, wash three times with 10% freezing glycerine by centrifugal results.Electricity consumption transforms above-mentioned DNA displacement box is transferred in the electroreception attitude E.coli MG1655 host cell, replaces target spot district on the karyomit(e) by regrouping process in the E.coli body.Screening box (CM-P in the said gene of the transduceing group
RhaB-Scel-sacB-1) the double-strand break reparation by the mediation of L-rhamnosyl inductive I-SceI endonuclease expression of enzymes cuts off from the reorganization E.coli bacterial strain that makes up.Use the fresh LB liquid nutrient medium that contains L-rhamnosyl and 5% sucrose to screen unmarked metathetical mutant then.
In sum, the invention provides a kind of method of karyomit(e) being carried out traceless modification.It should be noted that this method is applicable to the range gene transformation, the correlated condition in more than describing is non-limiting condition.All under enlightenment of the present invention, only do being equal to of correlated condition and replace or the formed technical scheme of equivalent transformation, all within the scope of protection of present invention.
Claims (10)
1. chromosomal traceless modification method, it is characterized in that: at first, make up comprise positive-selecting mark, anti-selection markers, endonuclease I-SceI, inducible promoter, I-SceI recognition site, with the reorganization box of target chromosome nucleotide sequence homologous target nucleic acid constant series; Then, the method for electricity consumption conversion comprises above-mentioned dna fragmentation input the host of recombinant plasmid; Then, repair phenomenon by the double-strand break of I-SceI mediation and from desirable recombinant chou, knock out following gene order: positive-selecting mark, inducible promoters, I-Scel gene, anti-selection markers; At last, the final recombinant chou of screening on sucrose medium.
2. chromosomal traceless modification method according to claim 1 is characterized in that: that described reorganization box transforms is Escherichia coli.
3. chromosomal traceless modification method according to claim 1 is characterized in that: the specific chromosomal region of modified microorganism according to the following steps,
1) prepares a linear DNA fragment, comprise and the λ-relevant homology arm B and C that can import targeted microorganisms of red reorganization, positive-selecting mark, anti-selection markers, the I-SceI gene, the I-SceI recognition site knocks out the relevant inducible promoters of selection markers, homologous fragment A with homologous recombination;
2) import linear DNA fragment to the microorganism that contains the described reorganization box of claim 1, by λ-Red reorganization replace the dna fragmentation homology arm and and homology arm homologous karyomit(e) between specific site;
3) in the conditioned medium of inducing I-SceI endonuclease expression of enzymes, cultivate specific chromosomal foci metathetical microorganism, cause double-strand break, homologous fragment A and and Segment A homologous chromosomal region between homologous recombination be used to knock out screening property mark, wherein homology arm B and the chromosomal 50-500bp homology that knocks out target spot one end, the 50-500bp homology of the homology arm C and the other end, homologous fragment A is the zone of an end 300-500bp of connection and homology arm B and homology arm C homologous chromosomal region.
4. chromosomal traceless modification method according to claim 3 is characterized in that: described step 1) and step 2) repeat repeatedly, prepare a plurality of different linear DNA fragments, with a series of specific chromosomal region of modified microorganism.
5. chromosomal traceless modification method according to claim 3 is characterized in that: described selection markers comprises positive-selecting mark and negative selection markers.
6. chromosomal traceless modification method according to claim 5, it is characterized in that: described positive-selecting mark is to screen from comprise visual certification mark, allow recombinant chou in the substratum that lacks nutrition source, to grow, give recombinant chou certain compound is produced resistance.
7. chromosomal traceless modification method according to claim 5 is characterized in that: described negative selection markers be a kind of in the presence of substrate pair cell the mark of lethal effect is arranged.
8. chromosomal traceless modification method according to claim 3 is characterized in that: described screening substrate comprises minimum medium, lacks one or more target cell or one or more essential nutrition sources of microbiotic.
9. chromosomal traceless modification method according to claim 1 is characterized in that: the modification of target chromosome is comprised that one or many ground " inserts one or more Nucleotide to the target cell genome " or " knocking out one or more Nucleotide from the target cell genome " or " one or more Nucleotide in the displacement target cell genome ".
10. chromosomal traceless modification method according to claim 9 is characterized in that: described target cell genome means nucleotide sequence or the non-genomic nucleotide sequence between native gene, native gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102132878A CN101892221A (en) | 2010-06-30 | 2010-06-30 | Traceless modification method of chromosome |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102132878A CN101892221A (en) | 2010-06-30 | 2010-06-30 | Traceless modification method of chromosome |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101892221A true CN101892221A (en) | 2010-11-24 |
Family
ID=43101567
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102132878A Pending CN101892221A (en) | 2010-06-30 | 2010-06-30 | Traceless modification method of chromosome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101892221A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559704A (en) * | 2010-12-23 | 2012-07-11 | 中国科学院上海生命科学研究院 | Method for knocking out gene in clostridium acetobutylicum |
| CN103451224A (en) * | 2013-08-26 | 2013-12-18 | 天津大学 | Traceless modification method of bacillus subtilis genome |
| CN104513829A (en) * | 2013-09-27 | 2015-04-15 | 中国科学院上海生命科学研究院 | Genetic modification method of target gene in genome |
| CN105505975A (en) * | 2016-01-06 | 2016-04-20 | 武汉康复得生物科技股份有限公司 | Bacillus gene traceless knockout/knockin plasmid and method, and kit |
| CN107109424A (en) * | 2014-12-01 | 2017-08-29 | 丹尼斯科美国公司 | Fungal host strain, DNA construct and application method |
| CN107236748A (en) * | 2017-07-28 | 2017-10-10 | 南通汇成生物科技有限公司 | A kind of recombinant plasmid, construction method and for the accurate genome manipulation of mycobacteria |
| CN108085328A (en) * | 2016-11-21 | 2018-05-29 | 中国科学院上海生命科学研究院 | A kind of method of DNA sequence dna editor |
| CN108676811A (en) * | 2018-05-28 | 2018-10-19 | 郝志敏 | A kind of seamless editor's carrier of gene and its application in organism gene editing |
| CN109385417A (en) * | 2017-08-03 | 2019-02-26 | 华东理工大学 | Internal DNA seamless integration method |
| CN112239765A (en) * | 2020-10-15 | 2021-01-19 | 天津科技大学 | Construction method of yeast fixed-point double-DSB synchronous induction model |
| WO2023046038A1 (en) * | 2021-09-24 | 2023-03-30 | Immunocan Biotech Co. Ltd. | Methods for large-size chromosomal transfer and modified chromosomes and organisims using same |
-
2010
- 2010-06-30 CN CN2010102132878A patent/CN101892221A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559704A (en) * | 2010-12-23 | 2012-07-11 | 中国科学院上海生命科学研究院 | Method for knocking out gene in clostridium acetobutylicum |
| CN102559704B (en) * | 2010-12-23 | 2014-08-27 | 中国科学院上海生命科学研究院 | Method for knocking out gene in clostridium acetobutylicum |
| CN103451224A (en) * | 2013-08-26 | 2013-12-18 | 天津大学 | Traceless modification method of bacillus subtilis genome |
| CN103451224B (en) * | 2013-08-26 | 2014-12-24 | 天津大学 | Traceless modification method of bacillus subtilis genome |
| CN104513829B (en) * | 2013-09-27 | 2018-03-06 | 中国科学院上海生命科学研究院 | The genetic modification method of target gene in genome |
| CN104513829A (en) * | 2013-09-27 | 2015-04-15 | 中国科学院上海生命科学研究院 | Genetic modification method of target gene in genome |
| CN107109424A (en) * | 2014-12-01 | 2017-08-29 | 丹尼斯科美国公司 | Fungal host strain, DNA construct and application method |
| CN105505975A (en) * | 2016-01-06 | 2016-04-20 | 武汉康复得生物科技股份有限公司 | Bacillus gene traceless knockout/knockin plasmid and method, and kit |
| CN108085328A (en) * | 2016-11-21 | 2018-05-29 | 中国科学院上海生命科学研究院 | A kind of method of DNA sequence dna editor |
| CN108085328B (en) * | 2016-11-21 | 2021-06-22 | 中国科学院分子植物科学卓越创新中心 | Method for editing DNA sequence |
| CN107236748A (en) * | 2017-07-28 | 2017-10-10 | 南通汇成生物科技有限公司 | A kind of recombinant plasmid, construction method and for the accurate genome manipulation of mycobacteria |
| CN107236748B (en) * | 2017-07-28 | 2020-12-11 | 南通汇成生物科技有限公司 | Recombinant plasmid, construction method and application in mycobacterium precise genome modification |
| CN109385417A (en) * | 2017-08-03 | 2019-02-26 | 华东理工大学 | Internal DNA seamless integration method |
| CN108676811A (en) * | 2018-05-28 | 2018-10-19 | 郝志敏 | A kind of seamless editor's carrier of gene and its application in organism gene editing |
| CN112239765A (en) * | 2020-10-15 | 2021-01-19 | 天津科技大学 | Construction method of yeast fixed-point double-DSB synchronous induction model |
| WO2023046038A1 (en) * | 2021-09-24 | 2023-03-30 | Immunocan Biotech Co. Ltd. | Methods for large-size chromosomal transfer and modified chromosomes and organisims using same |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101892221A (en) | Traceless modification method of chromosome | |
| US10301613B2 (en) | Targeted remodeling of prokaryotic genomes using CRISPR-nickases | |
| KR102623312B1 (en) | Enzyme with RUVC domain | |
| Kroth et al. | Genome editing in diatoms: achievements and goals | |
| EP2270180B1 (en) | DNA cloning vector plasmids and methods for their use | |
| US20220033791A1 (en) | Enzymes with ruvc domains | |
| TW201829773A (en) | Cas9 expression plasmid, gene editing system of escherichia coli and method thereof | |
| CN1427079A (en) | Construction method of multigene carrier and its application | |
| CN103725712B (en) | A kind of conditional gene knockout intermediate carrier without species restriction and its production and use | |
| CN103898140A (en) | Simple efficient gene editing method | |
| US7696335B2 (en) | Kits for multiple non-cross reacting recombination reactions utilizing loxP sequences | |
| Wang et al. | Targeted mutagenesis in hexaploid bread wheat using the TALEN and CRISPR/Cas systems | |
| US20070243616A1 (en) | In vivo alteration of cellular dna | |
| CN111386343A (en) | Methods for Kluyveromyces host cell genomic integration | |
| JP6867635B1 (en) | Genome modification method and genome modification kit | |
| Murata | Minichromosomes and artificial chromosomes in Arabidopsis | |
| CN115552002A (en) | Genome modification method and genome modification kit | |
| CN103468623B (en) | Indicator bacterium for gene engineering and application thereof | |
| CN111662932A (en) | Method for improving homologous recombination repair efficiency in CRISPR-Cas9 gene editing | |
| US20080286871A1 (en) | Modular genomes for synthetic biology and metabolic engineering | |
| CN115747242B (en) | Kit for eliminating plasmids, plasmid combination and gene editing, preparation method and application | |
| Sung et al. | Scarless chromosomal gene knockout methods | |
| Baek et al. | Positive selection improves the efficiency of DNA assembly | |
| CN117603898A (en) | Construction method and application of large-fragment DNA cross-species transfer auxiliary strain | |
| Swartjes et al. | Towards sequential conjugation-assisted laboratory evolution (SCALE) of Cas nucleases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20101124 |