CN105567718B - Construction method of vector for simultaneously expressing multiple sgRNAs - Google Patents
Construction method of vector for simultaneously expressing multiple sgRNAs Download PDFInfo
- Publication number
- CN105567718B CN105567718B CN201610044291.3A CN201610044291A CN105567718B CN 105567718 B CN105567718 B CN 105567718B CN 201610044291 A CN201610044291 A CN 201610044291A CN 105567718 B CN105567718 B CN 105567718B
- Authority
- CN
- China
- Prior art keywords
- sgrna
- vector
- mir
- sgrnas
- expression
- 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
Images
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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
-
- 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
Abstract
The invention relates to a construction method of a vector for simultaneously expressing multiple sgRNAs, wherein n sgRNAs to be connected in series are respectively inserted into corresponding 42230 vectors; amplifying the sgRNA by PCR2Then in the first sgRNA by seamless cloning1EcoR I site on expression vector enabling second sgRNA2Inserting an expression frame to obtain a vector for simultaneously expressing 2 sgRNAs; then, sgRNA is amplified through PCR amplification3Then 42230-sgRNA by seamless cloning1+2The EcoR I site at (A) realizes the third sgRNA3And inserting the expression frame to obtain a vector for simultaneously expressing 3 sgRNAs, and sequentially operating to obtain the sgRNAs. The invention can simultaneously knock out the gene family members, and solves the defects of the prior method.
Description
Technical Field
The invention belongs to the field of sgRNAs of a CRISPR/Cas9 system, and particularly relates to a construction method of a vector for simultaneously expressing multiple sgRNAs.
Background
CRISPR (clustered regulated short tandem repeats) originated from the immune system of bacteria and archaea, is currently the most widely used gene editing tool, and has been successfully applied to genome editing of species such as escherichia coli, yeast, mice, and humans.
CRISPR systems are divided into three categories, depending on how CRISPR directs RNA and CRISPR-binding nucleases in degrading target genes. Of these, Cas9(CRISRP-associated) is the most commonly used nuclease for type II (Patrick D.Hsu, Eric S.Lander, and Feng Zhang. development and Applications of CRISPR-Cas9for Genome engineering. cell 157,1263-1278 (2014)).
The identification and optimization of the important elements for the CRISPR to perform functions lay a foundation for the application of a CRISPR system. Jinek et al in vitro experiments demonstrated that the realization of CRISPR function relies on 4 important functional elements: tracrRNA, crRNA, Cas9 and PAM [ Martin Jinek, Krzysztoff Chylinski, Ines Fonfara, Michael Hauer, and Jennifer A. Doudna, Emmanuellel Charpentier. A Programmmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial immunity 337,816-821(2013) ]. Through further exploration by Cong L et al, a sgRNA (single guide RNA) capable of forming a secondary structure by itself, binding Cas9 and realizing site-specific cleavage and a human-derived codon-optimized Cas9 expression vector pX330-U6-Chimeric _ BB-CBh-hSpCas9(42230, addge) are successfully constructed [ Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F.multiple Genome Engineering CRISPR/systems Cas.science 339,819 823(2013) ]. From this point on, editing genomes using the CRISPR system becomes more flexible and convenient.
The advent of CRISPR simplifies the complex preparation of previous gene editing tools (ZFNs, TALENs). Because of the large amount of time and cost saved by such a convenient operation process, high-throughput gene editing using CRISPR is becoming a research hotspot. Originally Wang et al used 5 sgRNAs to transfect mouse embryonic stem cells simultaneously, and genotyping results showed that this method could achieve simultaneous editing of Multiple Genes [ Haoyi Wang, Hui Yang, Chikdu S.Shivalila, Meelad M.Dawlay, Albert W.Cheng, Feng Zhang, and Rudolf Jaenisch. one-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-media Genome engineering. cell.153,910-918(2013) ]. In order to improve the uniformity of the distribution of transfected sgrnas in cells, i.e., to achieve the editing of multiple genes simultaneously in the same cell, there have been successive studies focusing on the construction of vectors into which expression cassettes for multiple sgrnas are simultaneously inserted. Kabadi et al obtained 4 subclones containing sgRNA insertion sites by modifying existing sgRNA expression vectors, and both ends of sgRNA expression frames of these subclones respectively carried BsmBI enzyme cleavage sites. Due to the specificity of BsmBI enzyme recognition and cleavage sites, the ligation of 4 sgRNAs in order, i.e., the Golden Gate cloning method, can be realized by designing cohesive ends between the recognition sites and the cleavage sites, and they successfully realize the tandem expression of 4 sgRNAs [ Ami M.Kabadi, David G.Ousterout, Isaac B.Hilton and Charles A.Gersbach.polyplex CRISPR/Cas9-based genome engineering from a single viral vector.Nucleic Acids research.42(19) e147(2014) ]. The same as their construction idea, Sakuma et al transformed the existing 42230 plasmid to construct 7 subcloning vectors with sgRNA insertion sites: two Bsal enzyme cutting sites are inserted behind a sgRNA expression frame in the 1 st subcloning vector, different Basl enzyme cutting sites are respectively introduced at two ends of the sgRNA expression frame in other 6 vectors, a Golden Gate cloning method is also adopted, sticky ends generated by the base cutting of 7 subclones are connected end to end, and therefore the series connection of the sgRNAs in 7 is realized [ Tetsushi Sakuma, Ayami Nishikawa, Satoshi Kume, Kazuakii Chayama and Takashi Yamamoto.multiple genome engineering in human cells using all-in-one CRISPR/Cas9vector system.scientific Reports4:5400 (2014.) ]. Comparing existing methods for tandem expression of sgrnas based on Golden Gate, it can be seen that the number of tandem expression of sgrnas that can be achieved by these methods is limited by the number of subclones into which the sgrnas are inserted. Therefore, a scheme for achieving sgRNA expression in a more flexible way without relying on Golden Gate remains to be explored further.
Disclosure of Invention
The invention aims to provide a construction method of a vector capable of expressing multiple sgRNAs simultaneously, and provides a vector construction scheme capable of expressing multiple sgRNAs simultaneously in mammalian cells. In practical application, the sgrnas required by research can be flexibly connected in series according to the research requirement. In the research on the functions of the gene families, the scheme can simultaneously realize the simultaneous knockout of the members of the gene families; in the miRNA function research, two sgRNAs act on two ends of a coding region of the two sgRNAs simultaneously to knock out the whole miRNA. In conclusion, the present invention provides convenience for gene function studies.
The invention discloses a construction method of a vector for simultaneously expressing multiple sgRNAs, which comprises the following steps:
(1) respectively inserting n sgRNAs to be connected in series into corresponding 42230 vectors to obtain n sgRNAs expression vectors 42230-sgRNAsn(ii) a Wherein n is a positive integer of 1, 2, 3, 4 …;
(2) the Multi-sgRNA F1 and the Multi-sgRNA R were used as primers, and PCR amplification was performed to express 42230-sgRNA from the second sgRNA expression vector2Upper amplification of sgRNA2Then in the first sgRNA by seamless cloning1Expression vector 42230-sgRNA1The EcoR I site at (A) realizes the second sgRNA2Inserting an expression frame to obtain a recombinant vector 42230-sgRNA1+2；
(3) Using Multi-sgRNA F2 and Multi-sgRNA R as primers, PCR amplification was performed to express 42230-sgRNA from the third sgRNA expression vector3Upper amplification of sgRNA3Then 42230-sgRNA by seamless cloning1+2The EcoR I site at (A) realizes the third sgRNA3Inserting an expression frame to obtain a recombinant vector 42230-sgRNA1+2+3Then s isgRNA4The fourth sgRNA was achieved at the EcoR I site by seamless cloning4Inserting an expression frame, and sequentially operating to obtain a vector 42230-sgRNA for simultaneously expressing a plurality of sgRNAs1+2+3.。。+n(ii) a Wherein 42230-sgRNAnWherein all primers for amplifying the expression cassette with n & gt 2 are Multi-sgRNA F2 and Multi-sgRNA R as primers.
And (3) carrying out seamless cloning in the steps (2) and (3) by using a seamless cloning kit.
In the step (1), n is 4, and 4 sgRNA expression vectors are respectively: 42230-sgRNA1：42230-miR-505，42230-sgRNA2：42230-miR-29a，42230-sgRNA3：42230-miR-29c，42230-sgRNA4: 42230-SF 2. In the step (2)
Multi-sgRNA F1: AGGCAAAAAAGAAAAAGTAATTACGGTTCCTGGCCTTTTG; as shown in SEQ ID NO. 1;
Multi-sgRNA R:CGAGCTCTAGGAATTCTTTGTCTGCAGAATTGGCGC, as shown in SEQ ID NO. 2.
In the step (3)
Multi-sgRNA F2: GCGCCAATTCTGCAGACAAATTACGGTTCCTGGCCTTTTG; as shown in SEQ ID NO. 3;
Multi-sgRNA R:CGAGCTCTAGGAATTCTTTGTCTGCAGAATTGGCGC, as shown in SEQ ID NO. 4.
The Multi-sgRNA F2 and the Multi-sgRNA R can amplify all expression cassettes of 42230-sgRNA, n > 2.
And (3) carrying out colony PCR verification on the recombinant vectors in the steps (2) and (3), and then selecting a colony process which is positive in colony PCR for sequencing verification.
Carrying out colony PCR verification on the recombinant vector, and then selecting a colony process with positive colony PCR for sequencing verification, wherein the sequencing verification specifically comprises the following steps: the recombinant vector is transformed into DH5 alpha, after overnight culture, single clone is picked from a flat plate, colony PCR is carried out by taking Seq-F and Seq-R as primers to verify the insertion of sgRNA, then colony with positive colony PCR is selected, sequencing verification is carried out by taking Seq-R as a primer, and the sequence of the inserted sgRNA is detected in the recombinant plasmid as the correct plasmid of the colony.
The primer Seq-F: GATCGACCTGTCTCAGCTGG is shown as SEQ ID NO. 5; GCAACTAGAAGGCACAGTCG as shown in SEQ ID NO. 6.
Plasmids of colonies with correct sequencing verification results were extracted, and after transfection of 293A cells for 48 hours, the expression activity of sgRNA was verified by using a T7E1 enzyme identification method.
In the step (1), n sgrnas to be connected in series are respectively inserted into corresponding 42230 vectors to obtain n sgRNA expression vectors 42230-sgrnasn(ii) a Wherein n is a positive integer of 1, 2, 3, 4 …, and specifically:
sgrnas for N gene editing sites were designed according to the method provided by zhangfeng et al (references Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang f. multiple Genome Engineering CRISPR/Cas systems. science 339, 819-: the plus strand is a 20bp sequence which is formed by adding a PAM sequence (5 '-NGG-3') upstream of a 5 '-CACC-3' (a complementary sequence of a cohesive end of a first Bbs I enzyme cutting site of 42230) and starting with G, namely: 5 '-CACCGNNNNNNNNNNNNNNNNNNN-3'; the negative strand is a reverse complementary sequence of 5 '-AAAC-3' (the complementary sequence of the cohesive end of the second Bbs I enzyme cutting site of 42230) plus a 20bp sequence which is upstream of the PAM sequence (5 '-NGG-3') and is started by G, namely: 5 '-AAACNNNNNNNNNNNNNNNNNNNC-3'. Then, according to the method provided by the above steps, the synthesized positive and negative chains are denatured at 95 ℃ for 5min, annealed at room temperature for 10min to form a double-stranded DNA insert fragment, and under the catalytic action of T4DNA ligase, n sgRNAs to be connected in series are respectively inserted into 42230 vectors (http:// www.genome-engineering. org/criprpr) digested by Bbs I to obtain n sgRNA expression vectors 42230-sgRNAsn(ii) a Wherein n is a positive integer of 1, 2, 3, 4 ….
On the basis of a single sgRNA expression vector 42230, the invention only uses one vector 42230, two pairs of PCR primers consisting of three oligonucleotides, restriction enzymes EcoR I and Bbs I, a seamless cloning kit and a competent cell DH5 alpha, so as to realize the tandem expression of a plurality of sgRNAs on the same vector. Firstly, sgrnas to be connected in series are respectively inserted into corresponding 42230 vectors; then, amplifying an expression frame of the sgRNA2 from a second sgRNA expression vector by using a first pair of universal PCR amplification primers, and realizing the insertion of the second sgRNA expression frame at the EcoRI site of the first sgRNA expression vector through seamless cloning; then, using a second pair of universal PCR amplification primers to amplify an expression frame of the sgRNA3 from a third sgRNA expression vector, and realizing the insertion of the third sgRNA expression frame by seamlessly cloning an EcoR I site downstream of the second sgRNA expression frame; subsequent insertion of the sgRNA expression cassette completely repeats the third sgRNA insertion process. The invention always reserves the EcoRI I of the next sgRNA insertion site in the PCR amplification downstream primer, so the invention provides a flexible and open tandem expression scheme of a plurality of sgRNAs which is not limited by the number of subcloning vectors.
Advantageous effects
(1) The invention provides a vector construction scheme capable of simultaneously expressing multiple sgRNAs in mammalian cells. In practical application, the sgrnas required by research can be flexibly connected in series according to the research requirement. In the research on the functions of the gene families, the scheme can simultaneously realize the simultaneous knockout of the members of the gene families; in the miRNA function research, two sgRNAs act on two ends of a coding region of the two sgRNAs simultaneously to knock out the whole miRNA. In a word, the invention provides convenience for gene function research;
(2) unlike the Golden Gate based sgRNA tandem expression method, the present invention only relies on the existing sgRNA expression vector 42230 and seamless cloning kit, without the need to modify the vector into any subcloning vector; during primer design, the insertion of one sgRNA is ensured to always reserve an insertion site for the next sgRNA, so that the openness of the scheme in terms of the number of inserted sgRNAs is ensured; therefore, the scheme perfectly relieves the limitation of the number of the subcloning vectors on the number of the sgRNAs connected in series, and realizes the serial expression of a plurality of sgRNAs within the allowable range of the vector capacity.
Drawings
Fig. 1 is a diagram of a process for constructing a vector that simultaneously expresses multiple sgrnas;
FIG. 2 shows an electrophoretogram wherein (a) is an EcoR I linearization treatment 42230-miR-505 electrophoretogram; (b) an electrophoretogram of an insert sgRNA-miR-29a is subjected to PCR amplification from 42230-miR-29 a;
FIG. 3 is an electrophoretogram of colony PCR validation 42230-Multi-sgRNA-miR-505-miR-29 a; wherein a is an electrophoretogram for detecting whether the sgRNA-miR-29a is inserted or not by using colony PCR from number 1 to number 16, b is an electrophoretogram for detecting whether the sgRNA-miR-29a is inserted or not by using colony PCR from number 17 to number 32, and c is an electrophoretogram for detecting whether the sgRNA-miR-29a is inserted or not by using colony PCR from number 33 to number 40;
FIG. 4 is a sequencing validation graph of 42230-Multi-sgRNA-miR-505-miR-29 a;
FIG. 5 shows that the T7E1 enzyme verifies the activity of 42230-Multi-sgRNA-miR-505-miR-29a on two sgRNAs;
FIG. 6 is an electrophoretogram of 42230-multi-sgRNA-miR-505-miR-29a by EcoR I linearization treatment and an electrophoretogram of sgRNA-miR-29c of an insert PCR-amplified from 42230-miR-29 c;
FIG. 7 is a colony PCR validation 42230-Multi-sgRNA-miR-505-miR-29a-miR-29 c; wherein a is an electrophoresis diagram for detecting whether the sgRNA-miR-29c is inserted or not by using colony PCR from number 1 to number 16, b is an electrophoresis diagram for detecting whether the sgRNA-miR-29c is inserted or not by using colony PCR from number 17 to number 32, and c is an electrophoresis diagram for detecting whether the sgRNA-miR-29c is inserted or not by using colony PCR from number 33 to 41';
FIG. 8 is a sequencing validation graph of 42230-Multi-sgRNA-miR-505-miR-29a-miR-29 c;
FIG. 9 shows that the T7E1 enzyme verifies the activity of 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c in three sgRNA;
FIG. 10 is an electrophoretogram of 42230-multi-sgRNA-miR-505-miR-29a-miR-29c after EcoR I linearization treatment, and the right image is an electrophoretogram of sgRNA-SF2 of an insert PCR amplified from 42230-SF 2;
FIG. 11 is a colony PCR validation 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c-SF 2; wherein a is an electrophoretogram for detecting whether the sgRNA-SF2 is inserted or not by using colony PCR from 1 to 16, b is an electrophoretogram for detecting whether the sgRNA-SF2 is inserted or not by using colony PCR from 17 to 32, and c is an electrophoretogram for detecting whether the sgRNA-SF2 is inserted or not by using colony PCR from 33 to 40;
FIG. 12 is a sequencing validation graph of 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c-SF 2;
FIG. 13 shows that 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c-SF2 enzyme verifies four sgRNA activities by using T7E1 enzyme.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
Constructing a vector capable of expressing 4 sgRNAs simultaneously and respectively targeting 4 different genes
The first step is as follows: insert fragments are synthesized according to sequence information in the following table, and 4 sgRNA expression vectors 42230-miR-505, 42230-miR-29a, 42230-miR-29c and 42230-SF2 are constructed according to a sgRNA insertion method in 42230, and genes miR-505, miR-29a, miR-29c and SF2 are targeted respectively. Sequencing verified the insertion of each sgRNA.
Insert of Table 1.42230
The second step is that: inserting an expression frame of a second sgRNA-miR-29a into an EcoRI site of 42230-miR-505 to construct 42230-Multi-sgRNA-miR-505-miR-29 a:
(1) the 42230-miR-505 is linearized according to a reaction system and a program provided by the specification of a restriction enzyme EcoR I of NEB company, the linearization result of the vector is shown in figure 2a, and the vector after the linearization treatment of the EcoR I has a strip near 8500 bp;
(2) designing a first pair of PCR amplification primers, namely, Multi-sgRNA F1 (the first 20bp at the 5 'end is a homologous fragment at the upstream of an EcoR I site of 42230 to realize seamless cloning; the other 20bp is a sequence at the upstream of an expression frame of 42230sgRNA to realize amplification of the expression frame of the sgRNA by combining 42230), and Multi-sgRNA R (the first 16bp at the 5' end is a homologous fragment at the downstream of the EcoR I site of 42230, wherein the original EcoR I site of a 42230 vector is reserved to provide an enzyme cutting site for the insertion of the next sgRNA, the other 20bp is a sequence at the downstream of the expression frame of the 42230sgRNA to realize amplification of the expression frame of the sgRNA by combining 42230), and performing 50 mu l PCR amplification system: 5 XPCR buffer 10. mu.l, dNTP 4. mu.l, upstream and downstream primers 0.2. mu. lM 5. mu.l, DNA polymerase 1. mu.l, plasmid 2. mu.l, sterile double distilled water 32. mu.l, according to the PCR amplification program: denaturing at 98 ℃ for 10s, annealing at 60 ℃ for 15s, extending at 68 ℃ for 1min, and performing 35 cycles (the PCR conditions for amplification of sgRNA expression frames of sgRNA amplification are the same below), amplifying an expression frame of a second sgRNA-miR-29a from 42230-miR-29a, wherein the amplification result of the insert fragment is shown in FIG. 2b, and the length of the PCR amplification insert fragment is 501 bp;
Multi-sgRNA F1:AGGCAAAAAAGAAAAAGTAATTACGGTTCCTGGCCTTTTG
Multi-sgRNA R:CGAGCTCTAGGAATTCTTTGTCTGCAGAATTGGCGC
EcoR I cleavage site
(3) The ligation of the vector and the insert was achieved according to the reaction system and procedure provided in the instructions of the seamless cloning kit (near-shore protein science and technology limited, NR 001);
(4) transforming the recombinant vector into DH5 alpha, after overnight culture, picking single clone from a plate to perform colony PCR (PCR system: ddH2O 9.3.3 mu l, GT Buffer 1.5 mu l, dNTP 1.5 mu l, upstream and downstream primers (2 mu M)1.5 mu l, BSA 0.2 mu l, Taq enzyme 1 mu l, and finally picking single colony as a template. PCR program: pre-denaturation 95 ℃ 2min, thermal cycle: denaturation 95 ℃ 30s, annealing 60 ℃ 30s, extension 72 ℃ 1min, 40 cycles, and filling up 72 ℃ 10 min. the conditions for colony PCR are similar, wherein the conditions for the extension experiment are set as 30s x n according to the number of inserted sgRNA expression frames, the insertion of sgRNA-miR-29a is verified, the result of colony PCR is shown in figure 3, and the colony of PCR product size of 591bp is a positive clone;
Seq-F:GATCGACCTGTCTCAGCTGG
Seq-R:GCAACTAGAAGGCACAGTCG
(5) selecting a colony with positive colony PCR, performing sequencing verification by taking Seq-R as a primer, and detecting the sequence of sgRNA-miR-29a in recombinant plasmid 42230-Multi-sgRNA-miR-505-miR-29a by sequencing, wherein the sequencing result is shown in figure 4;
(6) plasmids of colonies with correct sequencing results were extracted, and after transfection of 293A cells for 48 hours, the expression activity of sgRNA was verified using the T7E1 enzyme identification method. The T7E1 enzyme digestion identification result is shown in FIG. 5, cells transfected with plasmid 42230-Multi-sgRNA-miR-505-miR-29a are subjected to T7E1 enzyme analysis, and a part of miR-505PCR products with the size of 770bp are cut into fragments with the sizes of about 500bp and 270 bp; meanwhile, a part of miR-29a PCR product with the size of 642bp is cut into fragments with the sizes of about 350bp and 290 bp; namely, the recombinant plasmid 42230-Multi-sgRNA-miR-505-miR-29a can realize gene editing at two sites.
The third step: inserting an expression frame of a third sgRNA-miR-29c into an EcoRI site of 42230-Multi-sgRNA-miR-505-miR-29a to construct 42230-Multi-sgRNA-miR-505-miR-29a-miR-29 c:
(1) according to the reaction system and the program provided by the specification of restriction enzyme EcoR I of NEB company, the linearization result of 42230-Multi-sgRNA-miR-505-miR-29a vector subjected to linearization treatment is shown in FIG. 6, and the vector subjected to linearization treatment by EcoR I has a band around 9000 bp;
(2) designing another PCR amplification upstream primer, namely, a Multi-sgRNA F2 (the first 20bp at the 5 'end is an EcoR I upstream homologous sequence in the vector constructed in the previous round, namely, a homologous sequence at the 3' end of 20bp of the Multi-sgRNA R to realize seamless cloning, the other 20bp is a sequence at the upstream of an 42230sgRNA expression frame, and combining 42230 to realize amplification of the sgRNA expression frame), and amplifying a Multi-sgRNA R (a downstream primer is always kept unchanged in the vector construction process, so that not only can the existence of the EcoRI at the site of the next sgRNA expression frame be ensured, but also the homologous sequence is recombined by the vector with constant front and back of the EcoR I, so that the Multi-sgRNA F2 and the Multi-sgRNA can be used as insertion fragment PCR amplification primers from the step to amplify the expression of a third sgRNA-miR-29c from 42230-miR-29c (the amplification conditions are as described in the previous round), the amplification result of the insert is shown in FIG. 6, and the length of the insert amplified by PCR is 501 bp;
Multi-sgRNA F2:GCGCCAATTCTGCAGACAAATTACGGTTCCTGGCCTTTTG；
Multi-sgRNA R:CGAGCTCTAGGAATTCTTTGTCTGCAGAATTGGCGC。
EcoR I cleavage site
(3) According to a reaction system and a program provided by the instructions of the seamless cloning kit, the connection of the vector and the insert fragment is realized;
(4) transforming the recombinant vector into DH5 alpha, after overnight culture, picking out a single clone from a plate to perform colony PCR (colony PCR conditions are as described above) by taking Seq-F and Seq-R as primers, verifying the insertion of sgRNA-miR-29c, wherein the colony PCR result is shown in figure 7, and a colony with a PCR product size of 1080bp is a positive clone;
(5) selecting a colony with positive colony PCR, performing sequencing verification by taking Seq-R as a primer, and detecting the sequence of sgRNA-miR-29c in recombinant plasmid 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c through sequencing, wherein the sequencing result is shown in figure 8;
(6) plasmids of colonies with correct sequencing results were extracted, and after transfection of 293A cells for 48 hours, the expression activity of sgRNA was verified using the T7E1 enzyme identification method. The T7E1 enzyme digestion identification result is shown in FIG. 9, cells transfected with plasmid 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c are subjected to T7E1 enzyme analysis, and a part of miR-505PCR products with the size of 770bp are cut into fragments with the sizes of about 500bp and 270 bp; a part of the 642bp miR-29a PCR product is cut into fragments with the sizes of about 350bp and 290 bp; part of the 801bp miR-29c PCR product is cut into fragments with sizes of about 470bp and 330bp, namely, the recombinant plasmid 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c can realize gene editing at three sites.
The fourth step: an expression frame of a fourth sgRNA-SF2 is inserted into an EcoRI site of 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c to construct 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c-SF2
(1) 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c is subjected to linearization treatment according to a reaction system and a program provided by the specification of a restriction enzyme EcoR I of NEB company, the linearization result of the vector is shown in FIG. 10, and a band appears near 9500bp on the vector subjected to the EcoR I linearization treatment;
(2) the expression cassette of the fourth sgRNA-SF2 was amplified from 42230-SF2 using multi-sgRNA F2 and multi-sgRNA R under the previously described amplification conditions, the amplification result of the insert was shown in FIG. 10, and the length of the PCR amplified insert was 501 bp;
(3) according to a reaction system and a program provided by the instructions of the seamless cloning kit, the connection of the vector and the insert fragment is realized;
(4) transforming the recombinant vector into DH5 alpha, after overnight culture, picking out a single clone from a plate, carrying out colony PCR (colony PCR conditions are as described above) by using Seq-F and Seq-R as primers, verifying the insertion of sgRNA-SF2, wherein the result of colony PCR is shown in figure 11, and a colony with the PCR product size of 1545bp is a positive clone;
(5) selecting a colony with positive colony PCR, performing sequencing verification by taking Seq-R as a primer, and detecting the sequence of sgRNA-SF2 in recombinant plasmid 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c-SF2 by sequencing, wherein the sequencing result is shown in figure 12;
(6) plasmids of colonies with correct sequencing results were extracted, and after transfection of 293A cells for 48 hours, the expression activity of sgRNA was verified using the T7E1 enzyme identification method. The T7E1 enzyme digestion identification result is shown in FIG. 13, cells transfected with plasmid 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c are subjected to T7E1 enzyme analysis, and a part of miR-505PCR products with the size of 770bp are cut into fragments with the sizes of about 500bp and 270 bp; a part of the 642bp miR-29a PCR product is cut into fragments with the sizes of about 350bp and 290 bp; part of the 801bp miR-29c PCR product is cut into fragments with the sizes of about 470bp and 330bp, and part of the 500bp SF2PCR product is cut into fragments with the sizes of about 300bp and 200bp, namely, the recombinant plasmid 42230-Multi-sgRNA-miR-505-miR-29a-miR-29c-SF2 can realize gene editing at four sites.
Claims (6)
1. A construction method of a vector for simultaneously expressing multiple sgRNAs comprises the following steps:
(1) will be provided withRespectively inserting n sgRNAs to be connected in series into corresponding 42230 vectors to obtain n sgRNAs expression vectors 42230-sgRNAsn；
(2) The Multi-sgRNA F1 and the Multi-sgRNA R were used as primers, and PCR amplification was performed to express 42230-sgRNA from the second sgRNA expression vector2Upper amplification of sgRNA2Then in the first sgRNA by seamless cloning1Expression vector 42230-sgRNA1The EcoR I site at (A) realizes the second sgRNA2Inserting an expression frame to obtain a recombinant vector 42230-sgRNA1+2；
Wherein Multi-sgRNA F1: AGGCAAAAAAGAAAAAGTAATTACGGTTCCTGGCCTTTTG; Multi-sgRNA R: CGAGCTCTAGGAATTCTTTGTCTGCAGAATTGGCGC；
(3) Using Multi-sgRNA F2 and Multi-sgRNA as primers, PCR amplification was performed to express 42230-sgRNA from the third sgRNA expression vector3Upper amplification of sgRNA3Then 42230-sgRNA by seamless cloning1+2The EcoR I site at (A) realizes the third sgRNA3Inserting an expression frame to obtain a recombinant vector 42230-sgRNA1+2+3Then, the amplified sgRNA is amplified by using the same pair of primers4The fourth sgRNA was achieved at the EcoR I site by seamless cloning4Inserting an expression frame, and sequentially operating to obtain a vector 42230-sgRNA for simultaneously expressing a plurality of sgRNAs1+2+3.。。+n(ii) a Wherein n is 2, 3 or 4;
wherein Multi-sgRNA F2: GCGCCAATTCTGCAGACAAATTACGGTTCCTGGCCTTTTG; Multi-sgRNA R: CGAGCTCTAGGAATTCTTTGTCTGCAGAATTGGCGC。
2. The method for constructing a vector for simultaneously expressing multiple sgrnas according to claim 1, wherein: and (3) carrying out seamless cloning in the steps (2) and (3) by using a seamless cloning kit.
3. The method for constructing a vector for simultaneously expressing multiple sgrnas according to claim 1, wherein: in the step (1), n is 4, and 4 sgRNA expression vectors are respectively: 42230-sgRNA1：42230-miR-505，42230-sgRNA2：42230-miR-29a，42230-sgRNA3：42230-miR-29c，42230-sgRNA4: 42230-SF 2; wherein 42230-SF2-F is CACCGTAGGTTACCCACGTAGATG; 42230-SF2-R is AAACCATCTACGTGGGTAACCTAC.
4. The method for constructing a vector for simultaneously expressing multiple sgrnas according to claim 1, wherein: and (3) carrying out colony PCR verification on the recombinant vectors in the steps (2) and (3), and then selecting a colony process which is positive in colony PCR for sequencing verification.
5. The method for constructing a vector for simultaneously expressing multiple sgrnas according to claim 4, wherein: carrying out colony PCR verification on the recombinant vector, and then selecting a colony process with positive colony PCR for sequencing verification, wherein the sequencing verification specifically comprises the following steps: transforming the recombinant vector into DH5 alpha, after overnight culture, selecting a single clone from a flat plate, carrying out colony PCR (polymerase chain reaction) verification on the insertion of sgRNAn by taking Seq-F and Seq-R as primers, then selecting a colony with positive colony PCR, carrying out sequencing verification by taking Seq-R as a primer, and detecting the plasmid with the correct sequence of the inserted sgRNAn in the recombinant plasmid by sequencing; wherein the primer Seq-F: GATCGACCTGTCTCAGCTGG; GCAACTAGAAGGCACAGTCG for Seq-R.
6. The method for constructing a vector for simultaneously expressing multiple sgrnas according to claim 5, wherein: plasmids of colonies with correct sequencing verification results were extracted, and after transfection of 293A cells for 48 hours, the expression activity of sgRNA was verified by using a T7E1 enzyme identification method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610044291.3A CN105567718B (en) | 2016-01-22 | 2016-01-22 | Construction method of vector for simultaneously expressing multiple sgRNAs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610044291.3A CN105567718B (en) | 2016-01-22 | 2016-01-22 | Construction method of vector for simultaneously expressing multiple sgRNAs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105567718A CN105567718A (en) | 2016-05-11 |
| CN105567718B true CN105567718B (en) | 2021-11-09 |
Family
ID=55878342
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610044291.3A Active CN105567718B (en) | 2016-01-22 | 2016-01-22 | Construction method of vector for simultaneously expressing multiple sgRNAs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105567718B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107964534A (en) * | 2016-10-19 | 2018-04-27 | 北京大学 | The method for transforming artificial liver seed cell |
| JP2021511776A (en) * | 2016-11-03 | 2021-05-13 | ユーヘルス バイオテック，リミテッド | Methods and compositions for cell reprogramming |
| 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 |
| CN110835635B (en) * | 2018-08-16 | 2024-01-09 | 深圳华大生命科学研究院 | Plasmid construction method for promoting expression of multiple tandem sgRNAs by different promoters |
| CN110885853A (en) * | 2018-09-11 | 2020-03-17 | 河南农业大学 | Method for constructing multi-sgRNA expression vector |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388456A (en) * | 2014-10-31 | 2015-03-04 | 东华大学 | Construction method of vector capable of simultaneously expressing two sgRNAs |
| CN104404036A (en) * | 2014-11-03 | 2015-03-11 | 赛业（苏州）生物科技有限公司 | Conditional gene knockout method based on CRISPR/Cas9 technology |
| US20150079680A1 (en) * | 2013-09-18 | 2015-03-19 | Kymab Limited | Methods, cells & organisms |
-
2016
- 2016-01-22 CN CN201610044291.3A patent/CN105567718B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20150079680A1 (en) * | 2013-09-18 | 2015-03-19 | Kymab Limited | Methods, cells & organisms |
| CN104388456A (en) * | 2014-10-31 | 2015-03-04 | 东华大学 | Construction method of vector capable of simultaneously expressing two sgRNAs |
| CN104404036A (en) * | 2014-11-03 | 2015-03-11 | 赛业（苏州）生物科技有限公司 | Conditional gene knockout method based on CRISPR/Cas9 technology |
Non-Patent Citations (8)
| Title |
|---|
| CＲISPＲ － Cas9 介导的基因组编辑技术的研究进展;郑小梅 等;《生物技术进展》;20151231;第5卷(第1期);第1-9页 * |
| CRISPR/Cas9 nuclease cleavage combined with Gibson assembly for seamless cloning;Jia-Wang Wang 等;《BioTechniques》;20151231;第58卷(第4期);第161-167页 * |
| CRISPR/Cas系统介导在哺乳动物细胞中mir-505基因的敲除;邓倩云;《中国优秀硕士学位论文全文数据库 基础科学辑》;20150715;第1-68页，尤其是第11页最后一段、第15页、第20-22页和第22页表2-2，第31-34页、第46-47页 * |
| Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system;Tetsushi Sakuma;《SCIENTIFIC REPORTS》;20140623;第1-6页，尤其是摘要部分、第2页Fig 1、第2页左栏最后一段到第3页最后一段、Methods部分 * |
| One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering;Haoyi Wang 等;《Cell》;20130509;第153卷;第910-918页 * |
| RNA-Guided Human Genome Engineering via Cas9;Prashant Mali 等;《Science》;20130215;第823-826页 * |
| Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems;Wei Li 等;《nature biotechnology》;20130831;第31卷(第8期);第684-686页 * |
| 一种多基因串联共表达载体的构建;何彰华 等;《中国生物工程杂志》;20111231;第31卷(第1期);第40-45页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105567718A (en) | 2016-05-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105567718B (en) | Construction method of vector for simultaneously expressing multiple sgRNAs | |
| CN111836894B (en) | Compositions for genome editing using CRISPR/Cpf1 systems and uses thereof | |
| US20240117330A1 (en) | Enzymes with ruvc domains | |
| WO2021023307A1 (en) | Crispr/cas9 gene editing system and application thereof | |
| US10913941B2 (en) | Enzymes with RuvC domains | |
| Li et al. | SWISS: multiplexed orthogonal genome editing in plants with a Cas9 nickase and engineered CRISPR RNA scaffolds | |
| CN107365786A (en) | A kind of method and its application being cloned into spacer sequences in CRISPR-Cas9 systems | |
| JP2021511824A (en) | Extended single guide RNA and its uses | |
| CN110144353B (en) | Rubber tree U6 gene promoter proHbU6.6 and cloning and application thereof | |
| CN110358767B (en) | Zymomonas mobilis genome editing method based on CRISPR-Cas12a system and application thereof | |
| CN110747187B (en) | Cas12a protein for identifying TTTV and TTV double-PAM sites, plant genome directed editing vector and method | |
| CN110551761B (en) | CRISPR/Sa-SepCas9 gene editing system and application thereof | |
| US11946039B2 (en) | Class II, type II CRISPR systems | |
| WO2022007959A1 (en) | System and method for editing nucleic acid | |
| US20230416710A1 (en) | Engineered and chimeric nucleases | |
| Cao et al. | The multiplexed CRISPR targeting platforms | |
| CN110577971B (en) | CRISPR/Sa-SauriCas9 gene editing system and application thereof | |
| CN110184268B (en) | Rubber tree U6 gene promoter proHbU6.2 and cloning and application thereof | |
| CN110551762B (en) | CRISPR/ShaCas9 gene editing system and application thereof | |
| US20220298494A1 (en) | Enzymes with ruvc domains | |
| CN104388456A (en) | Construction method of vector capable of simultaneously expressing two sgRNAs | |
| CN110144355B (en) | Rubber tree U6 gene promoter proHbU6.1 and cloning and application thereof | |
| CN110577969A (en) | CRISPR/Sa-SlugCas9 gene editing system and application thereof | |
| US20220220460A1 (en) | Enzymes with ruvc domains | |
| WO2022159742A1 (en) | Novel engineered and chimeric 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |