CN110835635B - Plasmid construction method for promoting expression of multiple tandem sgRNAs by different promoters - Google Patents
Plasmid construction method for promoting expression of multiple tandem sgRNAs by different promoters Download PDFInfo
- Publication number
- CN110835635B CN110835635B CN201810935546.4A CN201810935546A CN110835635B CN 110835635 B CN110835635 B CN 110835635B CN 201810935546 A CN201810935546 A CN 201810935546A CN 110835635 B CN110835635 B CN 110835635B
- Authority
- CN
- China
- Prior art keywords
- sequence
- restriction enzyme
- dna
- vector
- promoter
- 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.)
- Active
Links
- 108091027544 Subgenomic mRNA Proteins 0.000 title claims abstract description 55
- 239000013612 plasmid Substances 0.000 title claims abstract description 24
- 238000010276 construction Methods 0.000 title abstract description 24
- 230000001737 promoting effect Effects 0.000 title abstract description 4
- 239000013598 vector Substances 0.000 claims abstract description 129
- 239000012634 fragment Substances 0.000 claims abstract description 66
- 238000000034 method Methods 0.000 claims abstract description 53
- 108020004414 DNA Proteins 0.000 claims description 86
- 108091008146 restriction endonucleases Proteins 0.000 claims description 72
- 108091033409 CRISPR Proteins 0.000 claims description 31
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 29
- 125000006850 spacer group Chemical group 0.000 claims description 29
- 238000010367 cloning Methods 0.000 claims description 27
- 102000053602 DNA Human genes 0.000 claims description 24
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 24
- 238000010354 CRISPR gene editing Methods 0.000 claims description 23
- 238000011144 upstream manufacturing Methods 0.000 claims description 14
- 239000013604 expression vector Substances 0.000 claims description 12
- 238000005516 engineering process Methods 0.000 claims description 10
- 238000007857 nested PCR Methods 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 238000010362 genome editing Methods 0.000 abstract description 8
- 239000000047 product Substances 0.000 description 24
- 230000003321 amplification Effects 0.000 description 22
- 238000003199 nucleic acid amplification method Methods 0.000 description 22
- 238000003752 polymerase chain reaction Methods 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 101710116852 Molybdenum cofactor sulfurase 1 Proteins 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 108091028113 Trans-activating crRNA Proteins 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 1
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 101100112111 Caenorhabditis elegans cand-1 gene Proteins 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000005562 fading Methods 0.000 description 1
- IJJVMEJXYNJXOJ-UHFFFAOYSA-N fluquinconazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1N1C(=O)C2=CC(F)=CC=C2N=C1N1C=NC=N1 IJJVMEJXYNJXOJ-UHFFFAOYSA-N 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a plasmid construction method for promoting expression of a plurality of tandem sgRNAs by different promoters. The method adopts common vectors to be transformed into tool vectors, and then realizes rapid construction of two to multiple sgRNA tandem vectors through application of a plurality of universal sequences and universal primers. In the invention, because different sgRNAs are started by different promoters, the gene editing efficiency is not affected by series connection, and meanwhile, different target gene fragments are targeted, so that efficient and specific multi-gene editing is realized. In addition, the construction method is flexible, can replace different sgRNAs according to actual needs, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a plasmid construction method for promoting expression of a plurality of sgRNAs in series by different promoters.
Background
The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated) gene editing system is a third generation gene editing technology. Compared with the ZFN and TALEN of the first two generations of gene editing technology, CRISPR has higher cutting efficiency, simpler system and more free target site selection, so that the application of the CRISPR is wider and popular. CRISPR/Cas9 consists mainly of three parts, namely a 5 'end guide sequence crRNA (CRISPR-extended RNA) containing 20nt for targeted recognition, a tracrRNA (trans-activating RNA) complementarily paired with its 3' part, and a Cas9 nuclease. The crRNA and the tracrRNA mainly play a role in targeting recognition of DNA through nucleic acid complementary pairing, and Cas9 is used as helicase and nuclease to assist recognition and complete DNA site-directed cleavage. Wherein Ruvc and HNH elements of Cas9 cleave the non-complementary strand and complementary strand, respectively, causing DNA double strand breaks at specific locations. At present, the CRISPR/Cas9 system has been widely applied to targeted gene editing and modification of various organisms except bacteria, and meanwhile, the system is also applied to targeted treatment of malignant tumors, and researches show that the CRISPR system can cut specific mutations in the malignant tumors very efficiently and accurately so as to block the growth and metastasis of tumor cells.
In research exploration and future clinical applications, it is often the case that multiple genes need to be knocked out simultaneously. The current methods for targeting multiple gene loci are mainly two types, including simultaneous expression of multiple independent vectors, and multiple sgrnas are promoted by the same promoter: (1) One vector expresses one sgRNA while expressing multiple independent vectors; (2) Multiple sgrnas were ligated in tandem following the same promoter in the same vector. These two types of methods have the following disadvantages: (1) The main disadvantage of simultaneously expressing multiple independent vectors is that the use of multiple vectors reduces the efficiency with which multiple genes are simultaneously edited, and each time one vector is added, the efficiency with which multiple genes are simultaneously edited is proportionally reduced. (2) The disadvantage of multiple sgrnas being driven by the same promoter is that: first, sharing a single promoter for multiple sgrnas reduces the expression of each sgRNA, thereby reducing the efficiency with which multiple genes can be edited simultaneously; second, in terms of vector construction, once the tandem sgrnas are immobilized, the construction is time-consuming and laborious, replacing the new sgrnas. In summary, there is no very good rapid construction method for plasmids for polygene editing at present; the existing polygene editing technology is not high enough.
Disclosure of Invention
In order to effectively solve the technical problems, the invention adopts a common vector, and a plurality of sgrnas are quickly constructed in series by using a plurality of common sequences and common primers, and meanwhile, different sgrnas are started by different promoters, so that the gene editing efficiency is not affected by the series.
In a first aspect, the invention claims a method of constructing a vector for tandem expression of several sgrnas.
The method for constructing the vectors for serially expressing a plurality of sgrnas provided by the invention can be the following method A or method B:
method A: when the number of sgrnas is two, the method may comprise the steps of:
(A1) Constructing a tool carrier; the tool carrier contains DNA fragments; the DNA fragment comprises a promoter 1, a recognition sequence of type IIs restriction enzyme 1, a promoter 2 and a recognition sequence of type IIs restriction enzyme 2 from upstream to downstream.
Wherein the tool vector contains no part other than the type IIs restriction enzyme (including the type IIs restriction enzyme 1 and the type IIs restriction enzyme 2) on the DNA fragment.
(A2) Based on Golden Gate assembly technology, cloning a DNA sequence for expressing a spacer sequence (spacer) on sgRNA1 (i.e. a DNA sequence for expressing a target sequence in crRNA in sgRNA1 for identifying a target gene to be mutated) and a DNA sequence for expressing a spacer sequence on sgRNA2 into the tool vector by using the type IIs restriction enzyme 1 and the type IIs restriction enzyme 2, respectively, to the recognition sequence of the type IIs restriction enzyme 1 and the recognition sequence of the type IIs restriction enzyme 2, thereby obtaining an intermediate vector.
(A3) Cloning the functional fragment on the intermediate vector into a CRISPR plasmid to obtain a vector for tandem expression of two sgRNAs.
The functional fragment is a DNA fragment which sequentially contains the following elements from the upstream to the downstream on the intermediate vector: said promoter 1, said DNA sequence for expressing a spacer on sgRNA1, said promoter 2 and said DNA sequence for expressing a spacer on sgRNA 2.
Wherein the cloning of the functional fragment into the CRISPR plasmid may be seamless cloning of the functional fragment into the CRISPR plasmid in an overlap extension PCR manner.
Method B: when the number of sgrnas is three or more, the method may comprise the steps of:
(B1) Constructing a tool carrier A and a tool carrier B;
the tool carrier A contains a DNA fragment A; the DNA fragment A sequentially comprises a recognition sequence of a promoter A1 and a recognition sequence of An IIs type restriction enzyme A1, a recognition sequence of a promoter A2 and a recognition sequence of An IIs type restriction enzyme A2, a … … and a recognition sequence of a promoter An and An IIs type restriction enzyme An from upstream to downstream; n is an integer greater than or equal to 2 (e.g., n is 2).
The tool carrier B contains a DNA fragment B; the DNA fragment B sequentially comprises a recognition sequence of a promoter B1 and a recognition sequence of an IIs type restriction enzyme B1, a recognition sequence of a promoter B2 and a recognition sequence of an IIs type restriction enzyme B2, a … … recognition sequence of a promoter Bm and a recognition sequence of an IIs type restriction enzyme Bm from upstream to downstream; m is an integer greater than or equal to 2 (e.g., m is 2).
Wherein the tool vector (including the tool vector A and the tool vector B) contains no part except the corresponding type IIs restriction enzymes (including the type IIs restriction enzymes A1, A2, … …, an and the type IIs restriction enzymes B1, B2, … … and Bm) on the corresponding DNA fragments (including the DNA fragment A and the DNA fragment B).
(B2) Based on Golden Gate splicing technology, the IIs type restriction enzyme A1, the IIs type restriction enzymes A2 and … … and the IIs type restriction enzyme An are adopted to clone a DNA sequence for expressing a spacer sequence on sgRNa-A1, a DNA sequence for expressing a spacer sequence on sgRNa-A2, … … and a DNA sequence for expressing a spacer sequence on sgRNa-An into the recognition sequence of the IIs type restriction enzyme A1, the recognition sequence of the IIs type restriction enzyme A2, … … and the recognition sequence of the IIs type restriction enzyme An in the tool carrier A respectively to obtain An intermediate carrier A.
Based on Golden Gate splicing technology, the IIs type restriction enzyme B1, the IIs type restriction enzymes B2 and … … and the IIs type restriction enzyme Bm are adopted to clone a DNA sequence for expressing a spacer sequence on the sgRNA-B1, a DNA sequence for expressing a spacer sequence on the sgRNA-B2, a … … and a DNA sequence for expressing a spacer sequence on the sgRNA-Bm into the recognition sequence of the IIs type restriction enzyme B1, the recognition sequence of the IIs type restriction enzyme B2, … … and the recognition sequence of the IIs type restriction enzyme Bm in the tool carrier B respectively, so as to obtain an intermediate carrier B.
(B3) And seamlessly cloning the functional fragment A on the intermediate vector A and the functional fragment B on the intermediate vector B into a CRISPR plasmid in an overlapping extension PCR mode to obtain a plurality of vectors for serially expressing sgRNAs.
The functional fragment A is 1 to n DNA fragments which are to be expressed and are positioned on the intermediate vector A and have the following structural units: a promoter Ax, a DNA sequence for expressing a spacer sequence on sgRNa-Ax; x is an integer from 1 to n.
The functional fragment B is 1 to m DNA fragments which are to be expressed and are positioned on the intermediate vector B and have the following structural units: a promoter Ay, a DNA sequence for expressing a spacer sequence on sgRNa-Ay; y is an integer from 1 to m.
The total number of the structural units contained on the functional fragment A and the functional fragment B is three or more (i.e., the resulting vector expresses three or more tandem sgRNAs).
Further, in the step (B3) of the method B, when the overlap extension PCR is performed, the portion of the primer used that matches the intermediate vector A or/and the intermediate vector B is a universal sequence designed for a portion of the intermediate vector A or/and the intermediate vector B other than the structure "DNA sequence for expression of spacer sequence on sgRNA".
Further, in step (B3) of the method B, when the overlap extension PCR is performed, a portion of the primer used that matches the CRISPR plasmid is a universal sequence designed for a site to be inserted of the CRISPR plasmid.
Further, each promoter (e.g., the promoter 1, the promoter A1, the promoter B1, etc.) in the method may be selected from the following: hU6 promoter, mU6 promoter, h7SK promoter, hH1 promoter, etc.
Further, each type IIs restriction enzyme (e.g., type IIs restriction enzyme 1, type IIs restriction enzyme A1, type IIs restriction enzyme B1, etc.) in the method may be selected from the following: bbsI, bsmBI, etc.
Further, in the method a, the DNA fragment may be the DNA fragment a or the DNA fragment B in the method B. In the method B, the DNA fragment A may contain, in order from upstream to downstream, an hU6 promoter, a recognition sequence for type IIs restriction enzyme BbsI, an mU6 promoter and a recognition sequence for type IIs restriction enzyme BsmBI (denoted as-hU 6-BbsI-mU 6-BsmBI); the DNA fragment B may contain, in order from upstream to downstream, the h7SK promoter, the recognition sequence for the type IIs restriction enzyme BbsI, the hH1 promoter and the recognition sequence for the type IIs restriction enzyme BsmBI (denoted-h 7 SK-BbsI-hH 1-BsmBI) - (n and m are both 2).
Still further, in the method a, the tool carrier may be the tool carrier a or the tool carrier B in the method B. In the method B, the tool vector a may be a recombinant vector obtained by inserting the DNA fragment a into a pcs2+ vector at a multiple cloning site; the tool vector B may be a recombinant vector obtained by inserting the DNA fragment B into a pCS2+ vector at a multiple cloning site.
More specifically, the sequence of the DNA fragment A (-hU 6-BbsI-mU 6-BsmBI-) is specifically shown in SEQ ID No. 1; the sequence of the DNA fragment B (-h 7 SK-BbsI-hH 1-BsmBI-) is specifically shown in SEQ ID No. 2. Wherein, the 41 st to 281 th positions of SEQ ID No.1 are hU6 promoter sequences, and the 486 th to 740 th positions are mU6 promoter sequences. The 84 th to 326 th positions of SEQ ID No.2 are h7SK promoter sequence, and the 540 th to 754 th positions are hH1 promoter sequence. Correspondingly, the tool vector A is specifically a recombinant vector (named pCS2+ -hU 6-BbsI-mU 6-BsmBI) obtained by replacing the 79 th-120 th position (namely MCS 1) of the pCS2+ vector with the sequence shown in SEQ ID No. 1; the tool vector B is specifically a recombinant vector (named pCS2+ -h 7 SK-BbsI-hH 1-BsmBI) obtained by replacing the 79 th-120 th position (namely MCS 1) of the pCS2+ vector with the sequence shown in SEQ ID No. 2.
Further, the CRISPR plasmid may be a Lenkicrispr V2 vector. CRISPR plasmid systems fall into two categories, one is a single plasmid system, which contains both Cas9 protein and sgRNA, lentiCRISPR V2 vector; and secondly, a double-plasmid system, wherein one plasmid expresses Cas9 protein, and the other plasmid carries sgRNA. Whichever type may be used herein, the choice of which may be desired is based on different needs.
In the method, in the step (B3) of the method B, the primer used in performing the overlap extension PCR may be selected from the group consisting of:
(1) P1-F: single stranded DNA shown in SEQ ID No. 3;
(2) P2-R: single stranded DNA shown in SEQ ID No. 4;
(3) P3-F: single stranded DNA shown in SEQ ID No. 5;
(4) P4-R: single stranded DNA shown in SEQ ID No. 6;
(5) P5-F: single stranded DNA shown in SEQ ID No. 7;
(6) P6-R: single stranded DNA shown in SEQ ID No. 8;
(7) P7-F: single stranded DNA shown in SEQ ID No. 9;
(8) P8-R: single stranded DNA shown in SEQ ID No. 10;
(9) P9-R: single stranded DNA shown in SEQ ID No. 11;
(10) P10-F: single stranded DNA shown in SEQ ID No. 12;
(11) P11-R: single stranded DNA shown in SEQ ID No. 13;
(12) P12-F: single stranded DNA shown in SEQ ID No. 14.
In a specific embodiment of the present invention, the tool vector A is specifically the pCS2+ -hU 6-BbsI-mU 6-BsmBI-vector described above, and the tool vector B is specifically the pCS2+ -h 7 SK-BbsI-hH 1-BsmBI-vector described above. The CRISPR plasmid is specifically a LeniCRISPR V2 vector.
Accordingly, when constructing a vector for tandem expression of two sgrnas, the overlap extension PCR is performed as follows (a 1) or (a 2):
(a1) Carrying out PCR amplification by taking the tool carrier A as a template and taking the P1-F and the P2-R as primers to obtain an amplification product 1; carrying out PCR (polymerase chain reaction) amplification by taking a LeniCRISPR V2 vector as a template and taking the P3-F and the P4-R as primers to obtain an amplification product 2; the host bacteria are transformed seamlessly by the amplification product 1 and the amplification product 2, and the vectors (marked as expression vector 1) for serially expressing two sgRNAs are obtained from positive clones.
(a2) Carrying out PCR amplification by taking the tool carrier B as a template and taking the P5-F and the P6-R as primers to obtain an amplification product 3; carrying out PCR (polymerase chain reaction) amplification by taking a LeniCRISPR V2 vector as a template and taking the P7-F and the P8-R as primers to obtain an amplification product 4; the amplified product 3 and the amplified product 4 were transformed into host bacteria seamlessly, and a vector (designated as expression vector 2) for tandem expression of two sgrnas was obtained from the positive clone.
Accordingly, when constructing three vectors for sgRNA tandem expression, the overlap extension PCR was performed as follows: carrying out PCR (polymerase chain reaction) amplification by taking a LeniCRISPR V2 vector as a template and taking the P7-F and the P4-R as primers to obtain an amplification product 5; performing PCR amplification by using the expression vector 1 obtained in the step (a 1) as a template and the P1-F and the P9-R as primers to obtain an amplification product 6; performing PCR amplification by using the expression vector 2 obtained in the step (a 2) as a template and the P10-F and the P6-R as primers to obtain an amplification product 7; the host bacteria are transformed seamlessly by the amplification product 5, the amplification product 6 and the amplification product 7, and three vectors (marked as expression vectors 3) for serially expressing sgRNAs are obtained from positive clones.
Accordingly, when constructing vectors for tandem expression of four sgrnas, the overlap extension PCR was performed as follows: carrying out PCR (polymerase chain reaction) amplification by taking a LeniCRISPR V2 vector as a template and taking the P7-F and the P4-R as primers to obtain an amplification product 5; performing PCR amplification by using the expression vector 1 obtained in the step (a 1) as a template and the P1-F and the P11-R as primers to obtain an amplification product 8; performing PCR amplification by using the expression vector 2 obtained in the step (a 2) as a template and the P12-F and the P6-R as primers to obtain an amplification product 9; the host bacteria are transformed seamlessly by the amplification product 5, the amplification product 8 and the amplification product 9, and four sgRNA tandem expression vectors (marked as expression vectors 4) are obtained from positive clones.
In a second aspect, the invention claims any of the following biomaterials:
(a1) A plurality of sgrnas expressed in tandem constructed by the method described in the first aspect;
(a2) A tool carrier, which is the tool carrier in the method a described in the first aspect;
(a3) A kit of parts consisting of said tool carrier a and said tool carrier B of the method B of the first aspect above;
(a4) A kit comprising a primer as described in the first aspect hereinbefore, and said tool carrier or said kit of parts.
In a third aspect, the invention claims the use of a tool vector as described in the second aspect above or of a kit of parts or of a primer as described in the preceding paragraph for constructing a vector for tandem expression of several sgrnas.
The core of the invention is that the common vector and the common sequence are utilized to quickly construct the sgRNA which contains two or more sgRNAs and is started by different promoters, and different target gene fragments are targeted at the same time, thereby realizing high-efficiency and specific polygene editing; meanwhile, the construction method is flexible, can replace different sgRNAs according to actual needs, and has wide application prospects.
Drawings
Fig. 1 is a schematic structural view of a tool carrier a and a tool carrier B.
FIG. 2 is a schematic diagram of the construction strategy of a vector for tandem expression of several sgRNAs in the present invention.
FIG. 3 is a comparison of V2 vector editing efficiency for two sgRNAs in tandem.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
pcs2+ vector: feng Hui organism, cat# QT080.
Lenigirspr V2 vector: addgene, #52961.
EXAMPLE 1 construction of vectors for tandem expression of several sgRNAs
1. Synthetic tool carrier
The invention selects pCS2+ vector as the original skeleton vector of the construction tool vector. The pCS2+ has no BbsI and BsmBI enzyme cutting sites, the length is only 2000 bases, the vector molecular weight is small, and the cloning is very convenient.
Different numbers of starter tool vectors with different promoter series can be selected and synthesized according to the requirement. The invention starts with two tool vectors (tool vector A and tool vector B), each with two different promoters linked thereto, the representation being shown in FIG. 1.
Wherein the tool carrier A contains a DNA fragment A (-hU 6-BbsI-mU 6-BsmBI), and the sequence of the DNA fragment A is specifically shown in SEQ ID No. 1. The 41 st to 281 th positions of SEQ ID No.1 are hU6 promoter sequence, and the 486 th to 740 th positions are mU6 promoter sequence. The tool vector A is a recombinant vector obtained by replacing the 79 th to 120 th positions (namely MCS 1) of the pCS2+ vector with the sequence shown in SEQ ID No.1, and is named pCS2+ -hU 6-BbsI-mU 6-BsmBI-.
The tool carrier B contains a DNA fragment B (-h 7 SK-BbsI-hH 1-BsmBI), and the sequence of the DNA fragment B is specifically shown in SEQ ID No. 2. The 84 th to 326 th positions of SEQ ID No.2 are h7SK promoter sequence, and the 540 th to 754 th positions are hH1 promoter sequence. The tool vector B is a recombinant vector obtained by replacing the 79 th to 120 th positions (namely MCS 1) of the pCS2+ vector with the sequence shown in SEQ ID No.2, and is named pCS2+ -h 7 SK-BbsI-hH 1-BsmBI-.
2. Construction strategy of vectors for serially expressing a plurality of sgRNAs
The construction strategy is schematically shown in fig. 2.
1. Construction of pCS2+ tool vector containing two sgRNAs
Primer sequences of sgRNA1 and sgRNA2 were designed and synthesized, and after primer annealing, they were ligated to hU6 and hH1 promoters of pCS2+ -hU 6-BbsI-mU 6-BsmBI-and pCS2+ -h 7 SK-BbsI-hH 1-BsmBI, respectively, using the corresponding type IIs restriction enzyme based on Golden Gate construction technique. Transforming, picking clone for sequencing identification, and obtaining the following two vectors:
①pCS2+—hU6-sgRNA1—mU6—
②pCS2+—h7SK—hH1-sgRNA2—
primer sequences of sgRNA3 and sgRNA4 are designed and synthesized, and after primer annealing, the primer sequences are respectively connected to mU6 and h7SK promoters of (1) pCS2+ -hU 6-sgRNA 1-mU 6-and (2) pCS2+ -h 7 SK-hH 1-sgRNA 2-vectors by adopting corresponding type IIs restriction endonucleases based on Golden Gate assembly technology. Transforming, picking clone for sequencing and identification to obtain a tool carrier containing two sgRNAs:
③pCS2+—hU6-sgRNA1—mU6-sgRNA3—
④pCS2+—h7SK-sgRNA4—hH1-sgRNA2—
2. seamless cloning construction of LeniCRISPR V2 vector containing two sgRNAs
(1) Construction of LeniCRISPR V2 vector containing two sgRNAs Using vector (3)
Takes a carrier (3) as a template
P1-F：5’-gttaattaaggtaccGAGGGCCTATTTCCCATGAT-3’(SEQ ID No.3)；
P2-R：5’-agcgaattcaaaaaaGCACCGACTCGGTGCCACTT-3’(SEQ ID No.4)。
PCR reaction system (50 μl): template 1.5 μl; 1.5. Mu.l of primer; 10 Xbuffer 5. Mu.l; mgSO (MgSO) 4 3μl；KOD-plus 1μl；dNTP 5μl；H 2 O was made up to 50. Mu.l.
The reaction procedure: 94 ℃ for 2min;98℃10s,56℃30s,68℃30s/kb,30 cycles; preserving at 16 ℃.
With Lenigirspr V2 as template
P3-F：5’-GCACCGAGTCGGTGCttttttgaattcgctagcta-3’(SEQ ID No.5)；
P4-R：5’-GGGAAATAGGCCCTCggtaccttaattaaccaaac-3’(SEQ ID No.6)。
PCR reaction system (50 μl): template 1.5 μl; 1.5. Mu.l of primer; 10 Xbuffer 5. Mu.l; mgSO (MgSO) 4 3μl；KOD-plus 1μl；dNTP 5μl；H 2 O was made up to 50. Mu.l.
The reaction procedure: 94 ℃ for 2min;98℃10s,56℃30s,68℃30s/kb,10 cycles; 98℃10s,60℃30s,68℃30s/kb,20 cycles; preserving at 16 ℃.
After the PCR reaction, the reaction products of the two PCRs are respectively subjected to gel cutting recovery, and then the gel recovery products are subjected to seamless cloning conversion, wherein the system is as follows: vector (50 ng-200 ng), fragment (20 ng-200 ng), 5 Xbuffer, exnase II, H 2 O was made up to 20. Mu.l. The experiment was set up without adding 5 Xbuffer and Exnase II and control.
After the isoclony grows out, identification is carried out, and the correct vector is obtained as follows:
⑤V2-hU6-sgRNA1—mU6-sgRNA3—
(2) Construction of LeniCRISPR V2 vector containing two sgRNAs Using vector (4)
Takes a carrier (4) as a template
P5-F：5’-gttaattaaggtaccTTTTGCTGGCCTTTTGCTCA-3’(SEQ ID No.7)；
P6-R：5’-agcgaattcaaaaaaGCACCGACTCGGTGCCACTT-3’(SEQ ID No.8)。
With Lenigirspr V2 as template
P7-F：5’-GCACCGAGTCGGTGC ttttttgaattcgctagcta-3’(SEQ ID No.9)；
P8-R：5’-AAAAGGCCAGCAAAAggtaccttaattaaccaaac-3’(SEQ ID No.10)。
The PCR reaction system and the conditions are the same as those in the step (1), seamless cloning transformation is carried out, cloning is carried out after cloning is grown, and identification is carried out, so that the obtained vector is as follows:
⑥V2—h7SK-sgRNA4—hH1-sgRNA2—
3. seamless cloning construction of LeniCRISPR V2 vector containing three sgRNAs
Cloning was performed in three sections:
with lenti CRISPR V2 as template
P7-F: the sequence is the same as above.
P4-R: the sequence is the same as above.
Takes a carrier (5) as a template
P1-F: the sequence is the same as above.
P9-R：5’-ATGACGTCAGCGTTCgcaccggatcaattgccgac-3’(SEQ ID No.11)。
Takes a carrier (6) as a template
P10-F：5’-caattgatccggtgcGAACGCTGACGTCATCAACC-3’(SEQ ID No.12)。
P6-R: the sequence is the same as above.
And three-step PCR to recover target fragments, link and transform. After cloning, the following vectors were obtained by identification:
⑦V2-hU6-sgRNA1—mU6-sgRNA3—hH1-sgRNA2—
4. seamless cloning construction of LeniCRISPR V2 vector containing four sgRNAs
Cloning was performed in three sections:
with lenti CRISPR V2 as template
P7-F: the sequence is the same as above.
P4-R: the sequence is the same as above.
Takes a carrier (5) as a template
P1-F: the sequences are the same as
P11-R：5’-TGCTAAATACTGCAGgcaccggatcaattgccgac-3’(SEQ ID No.13)。
Takes a carrier (6) as a template
P12-F：5’-caattgatccggtgcCTGCAGTATTTAGCATGCCC-3’(SEQ ID No.14)；
P6-R: the sequence is the same as above.
And three-step PCR to recover target fragments, link and transform. After cloning, the following vectors were obtained by identification:
⑧V2-hU6-sgRNA1—mU6-sgRNA3—h7SK-sgRNA4—hH1-sgRNA2—
3. application example of constructing vectors for tandem expression of a plurality of sgRNAs
Four target genes: PD-1, CTLA-4, SOAT-1, VISTA.
Wherein the sgRNA target sequence, i.e. the DNA sequence for expressing the spacer sequence (spacer) on the sgRNA1 (the DNA sequence for expressing the target sequence in the crRNA in the sgRNA1 for recognizing the target gene to be mutated) is specifically as follows:
sgPD-1:5'-GGCCAGGATGGTTCTTAGGT-3' (noted sgRNA 1);
sgCTLA-4:5'-GCACAAGGCTCAGCTGAACC-3' (noted sgRNA 2);
sgSOAT-1:5'-TTCCTACCGTTGTTTGGACC-3' (noted sgRNA 3);
sgVISTA:5'-AGACGTGGTACCGCAGCTCG-3' (designated as sgRNA 4).
By adopting the construction strategy of the step two, the following 4 sgRNA series expression vectors (confirmed by sequencing verification) are finally constructed:
(1)V2-hU6-sgRNA1—mU6-sgRNA3—
(2)V2-hU6-sgRNA2—mU6-sgRNA3—
(3)V2-h7SK-sgRNA4—hH1-sgRNA2—
(4)V2-h7SK-sgRNA4—hH1-sgRNA3—
the experiment was also set up with V2 vector expressing only one sgRNA as control.
Then, 293T cells were transfected with a V2 vector containing one sgRNA and two sgRNAs, respectively, and the puromycin-selected post-genome was subjected to T7E1 assay for gene editing efficiency. The method comprises the following steps:
primers were designed on the genome across the editing sites for sgRNA1, sgRNA2, sgRNA3 and sgRNA4, respectively, as follows:
primers for detection of sgRNA 1:
F：5’-CCCTTGTACTCCAGGAAATTCTCCA-3’；
R：5’-ACTTGTGAGCTCATCCTGAAACCCA-3’。
primers for detection of sgRNA 2:
F：5’-TCCAGCATCCAGGAGCACCCC-3’；
R：5’-GTGTTGGCAGCCTGGTGGCCTCCA-3’。
primers for detection of sgRNA 3:
F：5’-GTAATAAAATGCTCAGCACAGAATA-3’；
R：5’-GAGAAAAATATCACCAGCTCATCT-3’。
primers for detection of sgRNA 4:
F：5’-TTTCAGAAGTTCAAAGTCCAA-3’；
R：5’-CAGCATCGCCTTGGGTAAC-3’。
PCR is carried out on the edited cell genome DNA, and the PCR products are reacted according to the following proportion after gel recovery: PCR recovery of product x. Mu.l (200 ng); 10 XNEBuffer 2 2 μl; DNase/RNase free water was made up to 19. Mu.l.
The denaturation and fire-fading reaction is carried out in a PCR instrument under the following reaction conditions: 95 ℃ for 5min; 95-85-2 ℃/second, 85-25-0.1 ℃/second; preserving at 4 ℃.
Adding T7E1 enzyme for enzyme digestion: 19 μl of the post-fire product; t7Endonuclease I1. Mu.l.
The reaction time was 15min at 37℃and 1% agarose electrophoresis was performed. The cutting efficiency was judged from the relative intensity of the cut strip (small piece cut under each lane) and the original strip (original piece of each lane, i.e., the longest piece at the top).
The results are shown in FIG. 3. It can be seen that tandem connection of two sgrnas is comparable to the editing efficiency of a single sgRNA. This indicates that the sgrnas after tandem are equally efficient.
<110> Shenzhen China institute of great life science; hua Daji North Biotech Co.Ltd
<120> plasmid construction method for multiple tandem sgRNA expression by different promoters
<130> GNCLN181601
<160> 14
<170> PatentIn version 3.5
<210> 1
<211> 1007
<212> DNA
<213> Artificial sequence
<400> 1
ttttgctggc cttttgctca catgtaaggt cgggcaggaa gagggcctat ttcccatgat 60
tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 120
tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 180
gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 240
gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg ggtcttcgag 300
aagacctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt tatcaacttg 360
aaaaagtggc accgagtcgg tgcttttttt ctccgctgag cgtactgaac gccgcggtgg 420
agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca 480
tgatagatcc gacgccgcca tctctaggcc cgcgccggcc ccctcgcaca gacttgtggg 540
agaagctcgg ctactcccct gccccggtta atttgcatat aatatttcct agtaactata 600
gaggcttaat gtgcgataaa agacagataa tctgttcttt ttaatactag ctacatttta 660
catgataggc ttggatttct ataagagata caaatactaa attattattt taaaaaacag 720
cacaaaagga aactcaccct aactgtaaag taattgtgtg ttttgagact ataaatatcc 780
cttggagaaa agccttgttt aaaggacgaa acaccggaga cggtcgtctc tgttttagag 840
ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag 900
tcggtgcttt tttgttttag agctagaaat agcaagttaa aataaggcta gtccgttttt 960
agcgcgtgcg ccaattctgc agacaaatgg ctctagaggt acccgtt 1007
<210> 2
<211> 971
<212> DNA
<213> Artificial sequence
<400> 2
gcttggcgta atcatggtca ttttgctggc cttttgctca catgtgggta cctttaattc 60
tagtactatg catcgttcat tgtctgcagt atttagcatg ccccacccat ctgcaaggca 120
ttctggatag tgtcaaaaca gccggaaatc aagtccgttt atctcaaact ttagcatttt 180
gggaataaat gatatttgct atgctggtta aattagattt tagttaaatt tcctgctgaa 240
gctctagtac gataagcaac ttgacctaag tgtaaagttg agacttcctt caggtttata 300
tagcttgtgc gccgcttggg tacctcggga aaggacgaaa caccgggtct tcgagaagac 360
ctgttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa 420
gtggcaccga gtcggtgctt tttttctccg ctgagcgtac tgaacgccgc ggtggagctc 480
cagcttttgt tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatcctg 540
aacgctgacg tcatcaaccc gctccaagga atcgcgggcc cagtgtcact aggcgggaac 600
acccagcgcg cgtgcgccct ggcaggaaga tggctgtgag ggacagggga gtggcgccct 660
gcaatatttg catgtcgcta tgtgttctgg gaaatcacca taaacgtgaa atgtctttgg 720
atttgggaat cttataagtt ctgtatgaga ccactctttc ccagaaagga cgaaacaccg 780
gagacggtcg tctctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 840
tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag 900
ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag 960
aggtacccgt t 971
<210> 3
<211> 35
<212> DNA
<213> Artificial sequence
<400> 3
gttaattaag gtaccgaggg cctatttccc atgat 35
<210> 4
<211> 35
<212> DNA
<213> Artificial sequence
<400> 4
agcgaattca aaaaagcacc gactcggtgc cactt 35
<210> 5
<211> 35
<212> DNA
<213> Artificial sequence
<400> 5
gcaccgagtc ggtgcttttt tgaattcgct agcta 35
<210> 6
<211> 35
<212> DNA
<213> Artificial sequence
<400> 6
gggaaatagg ccctcggtac cttaattaac caaac 35
<210> 7
<211> 35
<212> DNA
<213> Artificial sequence
<400> 7
gttaattaag gtaccttttg ctggcctttt gctca 35
<210> 8
<211> 35
<212> DNA
<213> Artificial sequence
<400> 8
agcgaattca aaaaagcacc gactcggtgc cactt 35
<210> 9
<211> 35
<212> DNA
<213> Artificial sequence
<400> 9
gcaccgagtc ggtgcttttt tgaattcgct agcta 35
<210> 10
<211> 35
<212> DNA
<213> Artificial sequence
<400> 10
aaaaggccag caaaaggtac cttaattaac caaac 35
<210> 11
<211> 35
<212> DNA
<213> Artificial sequence
<400> 11
atgacgtcag cgttcgcacc ggatcaattg ccgac 35
<210> 12
<211> 35
<212> DNA
<213> Artificial sequence
<400> 12
caattgatcc ggtgcgaacg ctgacgtcat caacc 35
<210> 13
<211> 35
<212> DNA
<213> Artificial sequence
<400> 13
tgctaaatac tgcaggcacc ggatcaattg ccgac 35
<210> 14
<211> 35
<212> DNA
<213> Artificial sequence
<400> 14
caattgatcc ggtgcctgca gtatttagca tgccc 35
Claims (6)
1. A method for constructing a plurality of sgRNA tandem expression vectors is a method A or a method B as follows:
method A: when the plurality of sgrnas is two sgrnas, the method comprises the steps of:
(A1) Constructing a tool carrier; the tool carrier contains DNA fragments; the DNA fragment sequentially comprises a promoter 1, a recognition sequence of IIs type restriction enzyme 1, a promoter 2 and a recognition sequence of IIs type restriction enzyme 2 from upstream to downstream;
(A2) Based on Golden Gate splicing technology, cloning a DNA sequence for expressing a spacer sequence on sgRNA1 and a DNA sequence for expressing a spacer sequence on sgRNA2 into the tool vector by adopting the IIs type restriction enzyme 1 and the IIs type restriction enzyme 2 respectively to the recognition sequence of the IIs type restriction enzyme 1 and the recognition sequence of the IIs type restriction enzyme 2 to obtain an intermediate vector;
(A3) Cloning the functional fragment on the intermediate vector into a CRISPR plasmid to obtain a vector for tandem expression of two sgRNAs;
the functional fragment is a DNA fragment which sequentially contains the following elements from the upstream to the downstream on the intermediate vector: said promoter 1, said DNA sequence for expressing a spacer on sgRNA1, said promoter 2 and said DNA sequence for expressing a spacer on sgRNA 2;
in the method A, the DNA fragment comprises a hU6 promoter, a recognition sequence of type IIs restriction enzyme BbsI, a mU6 promoter and a recognition sequence of type IIs restriction enzyme BsmBI from upstream to downstream; alternatively, the DNA fragment comprises an h7SK promoter, a recognition sequence of type IIs restriction enzyme BbsI, an hH1 promoter and a recognition sequence of type IIs restriction enzyme BsmBI from upstream to downstream;
method B: when the number of sgrnas is three or more, the method comprises the steps of:
(B1) Constructing a tool carrier A and a tool carrier B;
the tool carrier A contains a DNA fragment A; the DNA fragment A sequentially comprises a recognition sequence of a promoter A1 and a recognition sequence of An IIs type restriction enzyme A1, a recognition sequence of a promoter A2 and a recognition sequence of An IIs type restriction enzyme A2, a … … and a recognition sequence of a promoter An and An IIs type restriction enzyme An from upstream to downstream; n is an integer of 2 or more;
the tool carrier B contains a DNA fragment B; the DNA fragment B sequentially comprises a recognition sequence of a promoter B1 and a recognition sequence of an IIs type restriction enzyme B1, a recognition sequence of a promoter B2 and a recognition sequence of an IIs type restriction enzyme B2, a … … recognition sequence of a promoter Bm and a recognition sequence of an IIs type restriction enzyme Bm from upstream to downstream; m is an integer greater than or equal to 2;
(B2) Based on Golden Gate splicing technology, cloning a DNA sequence for expressing a spacer sequence on sgRNa-A1, a DNA sequence for expressing a spacer sequence on sgRNa-A2, a … … and a DNA sequence for expressing a spacer sequence on sgRNa-An into the recognition sequence of the IIs type restriction enzyme A1, the recognition sequence of the IIs type restriction enzyme A2, … … and the recognition sequence of the IIs type restriction enzyme An in the tool vector A respectively by adopting the IIs type restriction enzyme A1, the IIs type restriction enzyme A2 and the IIs type restriction enzyme An to obtain An intermediate vector A;
based on Golden Gate splicing technology, cloning a DNA sequence for expressing a spacer sequence on sgRNA-B1, a DNA sequence for expressing a spacer sequence on sgRNA-B2, a … … DNA sequence for expressing a spacer sequence on sgRNA-Bm into the recognition sequence of the IIs type restriction enzyme B1, the recognition sequence of the IIs type restriction enzyme B2, … … and the recognition sequence of the IIs type restriction enzyme Bm in the tool carrier B respectively by adopting the IIs type restriction enzyme B1, the IIs type restriction enzyme B2 and the IIs type restriction enzyme Bm to obtain an intermediate carrier B;
(B3) Seamlessly cloning the functional fragment A on the intermediate vector A and the functional fragment B on the intermediate vector B into a CRISPR plasmid in an overlapping extension PCR mode to obtain a plurality of sgRNA tandem expression vectors;
the functional fragment A is 1 to n DNA fragments which are to be expressed and are positioned on the intermediate vector A and have the following structural units: a promoter Ax, a DNA sequence for expressing a spacer sequence on sgRNa-Ax; x is an integer from 1 to n;
the functional fragment B is 1 to m DNA fragments which are to be expressed and are positioned on the intermediate vector B and have the following structural units: a promoter Ay, a DNA sequence for expressing a spacer sequence on sgRNa-Ay; y is an integer from 1 to m;
in the method B, the DNA fragment A sequentially comprises an hU6 promoter, a recognition sequence of type IIs restriction enzyme BbsI, an mU6 promoter and a recognition sequence of type IIs restriction enzyme BsmBI from upstream to downstream; the DNA fragment B sequentially comprises an h7SK promoter, a recognition sequence of IIs type restriction enzyme BbsI, an hH1 promoter and a recognition sequence of IIs type restriction enzyme BsmBI from upstream to downstream;
in the method a and the method B, the CRISPR plasmid is a pentigrisr V2 vector;
in the method A, the sequence of the DNA fragment is shown in SEQ ID No.1 or SEQ ID No. 2;
in the method B, the sequence of the DNA fragment A is shown in SEQ ID No. 1; the sequence of the DNA fragment B is shown as SEQ ID No. 2.
2. The method according to claim 1, characterized in that: in the step (B3) of the method B, the portion of the primer used to match the intermediate vector A or/and the intermediate vector B in the overlap extension PCR is a universal sequence designed for a portion of the intermediate vector A or/and the intermediate vector B other than the structure "DNA sequence for expressing a spacer sequence on sgRNA".
3. The method according to claim 1 or 2, characterized in that: in step (B3) of method B, when the overlap extension PCR is performed, the portion of the primer used that matches the CRISPR plasmid is a universal sequence designed for the site of the CRISPR plasmid to be inserted.
4. The method according to claim 1, characterized in that: in the method A, the tool vector is a recombinant vector obtained by inserting the DNA fragment into a multi-cloning site of a pCS2+ vector;
in the method B, the tool vector A is a recombinant vector obtained by inserting the DNA fragment A into a multi-cloning site of a pCS2+ vector; the tool vector B is a recombinant vector obtained by inserting the DNA fragment B into a multi-cloning site of a pCS2+ vector.
5. The method according to claim 1, characterized in that: in step (B3) of method B, the primer used in performing the overlap extension PCR is selected from the group consisting of:
(1) P1-F: single stranded DNA shown in SEQ ID No. 3;
(2) P2-R: single stranded DNA shown in SEQ ID No. 4;
(3) P3-F: single stranded DNA shown in SEQ ID No. 5;
(4) P4-R: single stranded DNA shown in SEQ ID No. 6;
(5) P5-F: single stranded DNA shown in SEQ ID No. 7;
(6) P6-R: single stranded DNA shown in SEQ ID No. 8;
(7) P7-F: single stranded DNA shown in SEQ ID No. 9;
(8) P8-R: single stranded DNA shown in SEQ ID No. 10;
(9) P9-R: single stranded DNA shown in SEQ ID No. 11;
(10) P10-F: single stranded DNA shown in SEQ ID No. 12;
(11) P11-R: single stranded DNA shown in SEQ ID No. 13;
(12) P12-F: single stranded DNA shown in SEQ ID No. 14.
6. A vector for tandem expression of a plurality of sgrnas constructed by the method of any one of claims 1 to 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810935546.4A CN110835635B (en) | 2018-08-16 | 2018-08-16 | Plasmid construction method for promoting expression of multiple tandem sgRNAs by different promoters |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810935546.4A CN110835635B (en) | 2018-08-16 | 2018-08-16 | Plasmid construction method for promoting expression of multiple tandem sgRNAs by different promoters |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110835635A CN110835635A (en) | 2020-02-25 |
| CN110835635B true CN110835635B (en) | 2024-01-09 |
Family
ID=69573977
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810935546.4A Active CN110835635B (en) | 2018-08-16 | 2018-08-16 | Plasmid construction method for promoting expression of multiple tandem sgRNAs by different promoters |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110835635B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501170A (en) * | 2020-11-30 | 2021-03-16 | 武汉爱博泰克生物科技有限公司 | Method for constructing MLH1 gene knockout cell line |
| CN113980999A (en) * | 2021-01-21 | 2022-01-28 | 扬州大学 | CRISPR/Cas9 vector based on multi-site simultaneous knockout and construction method and application thereof |
| CN114250242A (en) * | 2021-12-29 | 2022-03-29 | 上海英基生物科技有限公司 | One-step BbsI enzyme digestion connection fragment assembly method, assembly kit and application |
| CN114438123B (en) * | 2022-03-07 | 2024-04-02 | 中量大黄山高质量发展研究院有限公司 | Dicotyledon polygene editing vector and construction method thereof |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002325573A (en) * | 2001-04-27 | 2002-11-12 | Japan Science & Technology Corp | Vector |
| CN104388456A (en) * | 2014-10-31 | 2015-03-04 | 东华大学 | Construction method of vector capable of simultaneously expressing two sgRNAs |
| CN105255937A (en) * | 2015-08-14 | 2016-01-20 | 西北农林科技大学 | Method for expression of CRISPR sgRNA by eukaryotic cell III-type promoter and use thereof |
| CN105567718A (en) * | 2016-01-22 | 2016-05-11 | 东华大学 | Building method of carrier for expressing multiple sgRNAs simultaneously |
| CN105861552A (en) * | 2016-04-25 | 2016-08-17 | 西北农林科技大学 | Establishment method of T7-RNA-polymerase-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing system |
| WO2016130697A1 (en) * | 2015-02-11 | 2016-08-18 | Memorial Sloan Kettering Cancer Center | Methods and kits for generating vectors that co-express multiple target molecules |
| CN106222186A (en) * | 2016-08-22 | 2016-12-14 | 河北省农林科学院粮油作物研究所 | The structure of entry vector pAmpGate and pGentGate and using method |
| CN106868031A (en) * | 2017-02-24 | 2017-06-20 | 北京大学 | A kind of cloning process of multiple sgRNA series parallels expression based on classification assembling and application |
| CN107043782A (en) * | 2017-04-10 | 2017-08-15 | 西南大学 | A kind of gene knockout method and its sgRNA fragments and application |
| CN107090466A (en) * | 2017-04-20 | 2017-08-25 | 清华大学 | Double sgRNA expression plasmids and its construction method in library |
| CN107326043A (en) * | 2017-07-18 | 2017-11-07 | 河北省农林科学院粮油作物研究所 | The structure and application method of a kind of multifunctional carrier |
| CN107475256A (en) * | 2017-08-01 | 2017-12-15 | 西南大学 | It is a kind of based on more target sequence sgRNA expression vectors of endogenous tRNA systems of processing and its application in plant gene editor |
-
2018
- 2018-08-16 CN CN201810935546.4A patent/CN110835635B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002325573A (en) * | 2001-04-27 | 2002-11-12 | Japan Science & Technology Corp | Vector |
| CN104388456A (en) * | 2014-10-31 | 2015-03-04 | 东华大学 | Construction method of vector capable of simultaneously expressing two sgRNAs |
| WO2016130697A1 (en) * | 2015-02-11 | 2016-08-18 | Memorial Sloan Kettering Cancer Center | Methods and kits for generating vectors that co-express multiple target molecules |
| CN105255937A (en) * | 2015-08-14 | 2016-01-20 | 西北农林科技大学 | Method for expression of CRISPR sgRNA by eukaryotic cell III-type promoter and use thereof |
| CN105567718A (en) * | 2016-01-22 | 2016-05-11 | 东华大学 | Building method of carrier for expressing multiple sgRNAs simultaneously |
| CN105861552A (en) * | 2016-04-25 | 2016-08-17 | 西北农林科技大学 | Establishment method of T7-RNA-polymerase-mediated CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing system |
| CN106222186A (en) * | 2016-08-22 | 2016-12-14 | 河北省农林科学院粮油作物研究所 | The structure of entry vector pAmpGate and pGentGate and using method |
| CN106868031A (en) * | 2017-02-24 | 2017-06-20 | 北京大学 | A kind of cloning process of multiple sgRNA series parallels expression based on classification assembling and application |
| CN107043782A (en) * | 2017-04-10 | 2017-08-15 | 西南大学 | A kind of gene knockout method and its sgRNA fragments and application |
| CN107090466A (en) * | 2017-04-20 | 2017-08-25 | 清华大学 | Double sgRNA expression plasmids and its construction method in library |
| CN107326043A (en) * | 2017-07-18 | 2017-11-07 | 河北省农林科学院粮油作物研究所 | The structure and application method of a kind of multifunctional carrier |
| CN107475256A (en) * | 2017-08-01 | 2017-12-15 | 西南大学 | It is a kind of based on more target sequence sgRNA expression vectors of endogenous tRNA systems of processing and its application in plant gene editor |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110835635A (en) | 2020-02-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110835635B (en) | Plasmid construction method for promoting expression of multiple tandem sgRNAs by different promoters | |
| CN107586835B (en) | Single-chain-linker-based construction method and application of next-generation sequencing library | |
| CN105646719B (en) | Efficient fixed-point transgenic tool and application thereof | |
| AU2005248371B2 (en) | Methods for dynamic vector assembly of DNA cloning vector plasmids | |
| CN110747187B (en) | Cas12a protein for identifying TTTV and TTV double-PAM sites, plant genome directed editing vector and method | |
| CN105821075A (en) | Establishment method of caffeine synthetase CRISPR/Cas9 genome editing vector | |
| EP2474619B1 (en) | Specific method for preparing joined dna fragments including sequences derived from target genes | |
| WO2015144045A1 (en) | Plasmid library comprising two random markers and use thereof in high throughput sequencing | |
| CN113337502B (en) | gRNA and its use | |
| CN101368188B (en) | Quick efficient plant manpower fine RNA expression vector construction method | |
| CN108424907B (en) | High-throughput DNA multi-site accurate base mutation method | |
| US20240301374A1 (en) | Systems and methods for transposing cargo nucleotide sequences | |
| CN110551762B (en) | CRISPR/ShaCas9 gene editing system and application thereof | |
| WO2023060539A1 (en) | Compositions and methods for detecting target cleavage sites of crispr/cas nucleases and dna translocation | |
| WO2021192596A1 (en) | Linked dna production method and vector combination for use therein | |
| JP2024509048A (en) | CRISPR-related transposon system and its usage | |
| JP2024509047A (en) | CRISPR-related transposon system and its usage | |
| CN111662932B (en) | Method for improving homologous recombination repair efficiency in CRISPR-Cas9 gene editing | |
| CN104774860B (en) | Construct, system and its application suitable for transformed cells | |
| CN114540356A (en) | Rhodosporidium toruloides promoter and application thereof | |
| CN104357438B (en) | DNA assembling and cloning method | |
| CN110551763B (en) | CRISPR/SlutCas9 gene editing system and application thereof | |
| CN114891791B (en) | sgRNA of specific targeting canine Rosa26 gene and application thereof | |
| KR102035654B1 (en) | Novel mutant insect cell line producing antimicrobial peptide or a preparation method thereof | |
| CN107312788A (en) | A kind of construction method of TALE repetitive sequences carrier |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |