CN104404036A - Conditional gene knockout method based on CRISPR/Cas9 technology - Google Patents
Conditional gene knockout method based on CRISPR/Cas9 technology Download PDFInfo
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Abstract
The invention discloses a primer used for gene knockout, which comprises five groups of primer, and can be respectively shown in a SEQ NO:1, a SEQ NO:2, a SEQ NO:3, a SEQ NO:4, a SEQ NO:5, a SEQ NO:6, a SEQ NO:7, a SEQ NO:8, SEQ NO:9 and a SEQ NO:10. The invention also discloses an application of the primer in gene knockout aspect. The invention also discloses a conditional gene knockout method based on a CRISPR/Cas9 technology. The method can performing condition specificity, space-time specificity, and medicine-induced type gene modification; harm due to other cells can be reduced, function of constitutive expression gene in a specific tissue can be researched; tissue and space specificity gene knockout or induction type gene knockout can be realized by only using Cas9 tool mice; test period is short, and time and cost are saved.
Description
Technical field
The invention belongs to genetic modification technical field, be specifically related to the conditional gene knockout method based on CRISPR/Cas9 technology.
Background technology
Progress along with science and technology and the continuous exploration to life science, people seem more urgent in vivo a certain gene in the research of particular organization, cell and the expression in the time.The site-specific recombination developed rapidly is in recent years the key gene operational tool adapting to this needs and produce, it can induce target gene inactivation in the duration of certain stages or in specific tissue, avoid target gene lack embryo's Deaths of causing or occur complex phenotypes, delete riddled basins, thus complete functional analysis is made to goal gene.The recombinase system be widely used at present has Cre-LoxP system, FLP-FRT system, R-RS system and I-SceI system, but applies maximum still Cre-LoxP systems.
Cre-LoxP recombinase system is that conditional gene is practiced shooting, induced gene is practiced shooting, the core technology of Space-time speciality gene targeting strategy.
Cre-LoxP systemic origin, in P1 phage, comprises following two kinds of compositions:
1. the DNA sequence dna of one section of 34bp, the core sequence of the inverted repeats containing two 13bp and a 8bp.This section of 34bp sequence is the recognition site of recombinase, is called as LoxP site.
2. Cre recombinase, it is a kind of monomeric protein be made up of 343 amino acid, can cause the DNA restructuring in LoxP site.The DNA of any sequence, when its between two LoxP sites time, under the effect of Cre recombinase or lacked (direction in two LoxP sites is identical), or direction is reversed (direction in two LoxP sites is contrary), as shown in Figure 1.
Utilize Cre-LoxP system to realize the knocking out under given conditions of certain specific gene in body, need two transgenic mices.First mouse: build a goal gene first in vitro, this fragment gene sequence built containing a LoxP site, proceeds in embryonic stem cell by its two ends respectively afterwards.Treated embryonic stem cell is implanted to the intrauterine of pseudopregnant mouse, makes it again bud into a complete embryo, finally becomes a transgenic mice.In this transgenic mice, LoxP site is introduced in the intron of corresponding gene, can not have an impact in theory to the function of corresponding gene, and therefore generally, the phenotype of this mouse is normal.Second transgenic mice: general ovocyte injection or the embryonic stem cell technologies of adopting obtains, and in this mouse, Cre recombinase is placed under the regulation and control of certain specific gene promotor, and it can be made to express under certain specific condition.Finally, allow these two mouse mating, containing above-mentioned two kinds of genotypic generation mices while generation will lack a certain specific gene in the cell of a certain particular type.
Artificial endonucleases Clustered Regularly Interspaced Short Palindromic Repeats, CRISPRs/Cas9 system, is extensively present in prokaryotic organism (most bacterium and all archeobacterias) genome.Since CRISPRs/Cas9 system was defined first from 2002, caused the common concern of various countries scientist with the structure of its uniqueness and special function always.CRISPR is roughly divided into 3 classes, for genetic modification is II type CRISPR system, its composition is comparatively simple, only needs Cas9 and two noncoding RNA:crRNA and trans-activation crRNA (tracrRNA), and three components can mediate the target degraded of exogenous dna fragment.In the operating process of reality, we only need to design specific sgRNA, and are transferred in the zygote of mouse together with Cas9 albumen.
Although Cre-loxp recombinase system can the conditionality of mediated gene knock out, avoid lacking the inferior position of traditional gene targetings such as embryo's premature death that functional gene causes, but exist a lot of uncertain:
1. there is implicit/false LoxP sequence in mammalian genes group, the conserved sequence of its sequence and LoxP may and incomplete same, but can be recombinated by the identification of Cre recombinase, and then to cause unnecessary DNA damage.
2. poor specificity, is difficult to the progeny mice obtaining existing double transgenic, success ratio <50%.
3. evaluation and screening process complexity, length consuming time.
Although existing CRISPR/Cas9 system is simple to operate, target accuracy is high, can not realize the specific knockdown of tissue, cell, space-time.
Summary of the invention
Goal of the invention: first technical problem to be solved by this invention there is provided a kind of primer for gene knockout.
Second technical problem to be solved by this invention there is provided the application of above-mentioned primer in gene knockout.
3rd technical problem to be solved by this invention there is provided a kind of conditional gene knockout method based on CRISPR/Cas9 technology.
The tissue specificity of this research knocks out technology, is by third generation genetic modification technology CRISPR/Cas9 and Cre-LoxP system globe area.The advantage of its existing CRISPR/Cas9 technology, can realize again the tissue specificity/drug induced gene knockout of gene; In addition again shorter than the cycle of Cre-LoxP technology, efficiency is high, decreases cytotoxicity.The present invention only needs to design specific sgRNA, can realize the specificity modification of the tissue of gene, space-time.Utilize the gene of the research constitutive expression that Cas9 instrument mouse technology can be efficient, special at short notice in a certain tissue or the function in the specific time, the research for human diseases provides theoretical foundation and animal model.This invention also can be used as clinical and supporting technology that is medical research, for the health of the mankind escorts in addition.
Technical scheme: in order to solve the problem, technical scheme of the present invention there is provided one group of primer for gene knockout, comprise following five groups of primers, first group of primer sequence is as shown in SEQ NO:1 and SEQ NO:2, second group of primer sequence is as shown in SEQ NO:3 and SEQ NO:4,3rd group of primer sequence is as shown in SEQ NO:5 and SEQ NO:6, and the 4th group of primer sequence is as shown in SEQ NO:7 and SEQ NO:8, and the 5th group of primer sequence is as shown in SEQ NO:9 and SEQNO:10.
The application of above-mentioned primer in gene knockout.
Based on a conditional gene knockout method for CRISPR/Cas9 technology, comprise the following steps:
1) structure of expression vector: build the sgRNA carrier of tissue specific expression and the Cas9 carrier of composing type/drug induced expression;
2), after expression vector order-checking correctly, electricity forwards in ES cell respectively; Verified by PCR or Southern Blot, obtain ES cell containing sgRNA expression plasmid respectively and containing the ES cell of Cas9 albumen and enlarged culturing;
3) by the Plastid transformation containing Cre recombinase in the ES cell containing Cas9 albumen, obtain and there is no the ES cell of the Cas9 expression vector of selection markers;
4) the ES cell containing Cas9 expression vector and the ES cell containing sgRNA expression plasmid are expelled in blastaea respectively;
5) blastaea is transplanted to respectively in the female mouse of replace-conceive, raises; The offspring produced is F
0for the Cas9 instrument mouse of the sgRNA mouse of tissue specific expression and composing type/drug induced;
6) by the correct Cas9 instrument mouse of detection and sgRNA mouse hybrid, tissue specificity/drug induced knock out mice is obtained.
Wherein, the sgRNA carrier of above-mentioned tissue specific expression comprises the promotor of tissue specific expression, Cre recombinase, reverse 2 LoxP sites and reverse U6 promotor, make it can only express Cre enzyme in particular organization, guide the U6 promotor inversion between LoxP site, thus start the expression of sgRNA, as shown in Figure 2.
Wherein, the sgRNA carrier of above-mentioned tissue specific expression comprises 2 identical LoxP sites of the promotor of tissue specific expression, Cre recombinase, the U6 promotor of forward, direction and termination area, it is made only in particular organization, to express Cre enzyme, knock out the termination area between two LoxP sites, thus start the expression framework of sgRNA.As shown in Figure 3.
Wherein, above-mentioned F
0construction process for tissue specific expression sgRNA mouse is as follows:
21) sgRNA fragment Design and synthesis: described sgRNA fragment sequence is as shown in SEQ NO:1 and SEQ NO:2; Described sgRNA fragment specific design is as follows:
A () finds the relevant information of target gene on Ensembl and NCBI, determine to knock out region;
B () knocks out after region is selected and utilizes Crispr software (http://crispr.mit.edu/) to design sgRNA fragment;
C () is analyzed the target spot that Crispr selects on ncbi database, select less sgRNA site, site of missing the target;
22) the sgRNA fragment of synthesis is annealed into double-stranded segment;
23) double-stranded segment is connected in linearized vector Church Cloning Vector or pCR-Blunt II-TOPO and obtains connecting product sgRNA expression plasmid;
24) connection product sgRNA expression plasmid is transformed, selects positive colony, carry out bacterium colony PCR checking;
25) product that bacterium colony PCR obtains is carried out agarose gel electrophoresis detection;
26) obtain verifying that correct clone carries out checking order the correct sgRNA expression plasmid that checks order;
27) sgRNA expression plasmid correct for order-checking electricity is gone in the ES cell of mouse; Verify through Southern Blot;
28) will verify that correct ES cell infusion is in the blastaea of mouse, be transferred to subsequently in the female mouse of replace-conceive, finally obtain F
0for the mouse of sgRNA.
Wherein, the construction process of the Cas9 instrument mouse of above-mentioned composing type/drug induced is as follows:
31) amplification of Cas9 albumen;
32) Cas9 albumen is connected the Cas9 carrier obtaining composing type/drug induced expression with Rosa26 carrier;
33) electricity after the Cas9 vector linearization of composing type/drug induced expression correct for order-checking is forwarded in ES cell;
34) positive ES cells containing Cas9 albumen is obtained with the Cas9 carrier of the Screening of Media composing type/drug induced expression containing Liu Suanyan NEOMYCIN SULPHATE; Subsequently with Southern Blot checking;
35) plasmid with Cre recombinase is forwarded in the positive ES cells containing Cas9 albumen, obtain the positive ES cells not having selection markers;
36) will the positive ES cells microinjection of selection markers be there is no in Mouse Blastocysts, be then transferred in the female mouse of replace-conceive, finally obtain the instrument mouse containing Cas9 albumen.
Wherein, the primer sequence that above-mentioned bacterium colony PCR adopts is as shown in SEQ NO:3 and SEQ NO:4, and the amplification condition of bacterium colony PCR is: denaturation 94 DEG C, 3min; Sex change 94 DEG C, 20s; Anneal 58 DEG C, 25s; Extend 72 DEG C, 20s, 25 circulations, extend 72 DEG C eventually, 5min.
Wherein, the amplification step of above-mentioned Cas9 albumen is as follows: the primer of design amplification Cas9 albumen, and its sequence is as shown in SEQNO:5 and SEQ NO:6; Adopt pcr amplification Cas9 albumen complete sequence, pcr amplification condition is: denaturation 98 DEG C, 3min; Sex change 98 DEG C, 25s; Anneal 63 DEG C, 25s; Extend 72 DEG C, 4min30s, 25 circulations, extend 72 DEG C eventually, 5min.
Wherein, above-mentioned Cas9 albumen and Rosa26 carrier Connection Step as follows: with Clontech company
the correct Cas9 sequence of detection is connected with through the linearizing carrier of AsisI and HpaI enzyme by HDClinging Kit test kit, after the vector intestinal bacteria connected, picked clones, bacterium colony PCR are verified, Suzhou Jin Weizhi company is sent to check order, what wherein bacterium colony PCR primer adopted is the primer shown in SEQ NO:7 and SEQ NO:8, the amplification condition of this bacterium colony PCR is: denaturation 95 DEG C, 3min; Sex change 95 DEG C, 30s; Anneal 58 DEG C, 30s; Extend 72 DEG C, 45s, 26 circulations, extend 72 DEG C eventually, 5min.
Cas9 albumen for prototype with Rosa26 carrier, is inserted into the Rosa26 region of mouse, avoids the cytotoxicity that the radom insertion of Cas9 albumen causes, as shown in Figure 4 by us.
Beneficial effect: the present invention, relative to prior art, has the following advantages: the advantage based on the conditional gene knockout technology of CRISPR/Cas9 technology:
1. simple to operate, high-level efficiency, low lethality rate, does not have species to limit.
2. condition specificity, Space-time speciality, drug induced genetic modification can be carried out.
3. the harm to other cells is reduced, the function of gene in particular organization of research constitutive expression; Avoid embryo's premature death that the disappearance of functional gene causes.
4. only need a Cas9 instrument mouse can realize tissue, Region-specificity gene knockout or inducible genes to knock out.
5. the test period is short, saves time and cost.
Accompanying drawing explanation
The conditional gene knockout mode chart of Fig. 1: Cre-LoxP recombinase system mediation; The DNA of any sequence, when it is between two LoxP sites, if direction, LoxP site is identical, then sequence deletion; If direction, LoxP site is contrary, then sequence reversing;
Fig. 2: the sgRNA expression vector mode chart of tissue specific expression; This carrier is made up of the expression framework of tissue-specific promoter, Cre recombinase, reverse LoxP site, reverse U6 promotor and sgRNA; This carrier only, in specific tissue, just can excite the expression of Cre recombinase, and then U6 promotor forward between the reverse LoxP site of mediation two start the expression that sgRNA expresses framework;
Fig. 3: the sgRNA expression vector mode chart of tissue specific expression; This carrier is expressed framework by tissue-specific promoter, Cre recombinase, U6 promotor, LoxP site, terminator and sgRNA and is formed; This carrier only, in specific tissue, just can excite the expression of Cre recombinase, and then knock out the termination area between two LoxP sites, thus starts the expression that sgRNA expresses framework;
Fig. 4: the Cas9 expression vector mode chart that broad spectrum is expressed; This carrier is with Rosa26 expression vector for framework, is made up of 5 ', the 3 ' homology arm of Rosa26, composing type/drug induced promotor, neo selection markers, LoxP site, Cas9 protein expression sequence, DTA termination area; Transfer them in Mice Body, homologous recombination technique will be utilized to be inserted into the Rosa26 region of mouse;
Fig. 5: the sgRNA expression vector of liver specific expression; This carrier is made up of the expression framework of the Alb promotor of liver specific expression, Cre recombinase, reverse LoxP site, reverse U6 promotor and sgRNA; This carrier, only in liver, just can excite the expression of Cre recombinase, and then U6 promotor forward between mediation two reverse LoxP sites start the expression that sgRNA expresses framework;
Fig. 6: the sgRNA bacterium colony PCR proof diagram be connected with carrier; The product size obtained is 338bp.DNA Marker stripe size is respectively 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp from bottom to top;
Fig. 7 ~ 8:sgRNA expression plasmid electricity goes in the ES cell of mouse, with Southern Blot the result figure; Genome after Xba I enzyme is cut, the figure after hybridizing with Alb probe, Cre and U6 probe, illustrate sgRNA complete proceeded in ES cell;
Fig. 9: Cas9 albumen amplification in vitro electrophorogram; The fragment of 3.9kb is obtained after pcr amplification; DNA Marker stripe size is respectively 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp, 2500bp, 3000bp, 3500bp, 4000bp, 5000bp, 6000bp, 8000bp, 10000bp from bottom to top;
Figure 10: the Cas9 protein expression vector mode chart that broad spectrum is expressed; This carrier is with Rosa26 expression vector for framework, is made up of 5 ', the 3 ' homology arm of Rosa26, CMV strong promoter, neo selection markers, LoxP site, Cas9 protein expression sequence, DTA termination area; Transfer them in Mice Body, homologous recombination technique will be utilized to be inserted into the Rosa26 region of mouse;
Figure 11: the sgRNA bacterium colony PCR proof diagram be connected with carrier; The product size obtained is 601bp; DNAMarker stripe size is respectively 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp from bottom to top;
Figure 12 ~ 13:Cas9 expression plasmid electricity goes in the ES cell of mouse, with Southern Blot the result figure; 3 ' end checking: after cutting with Hind II enzyme, wild-type fragment 9.3kb, saltant type 7.6kb; 5 ' end checking: after cutting with EcoR V enzyme, wild-type fragment 11.6kb, saltant type 5.2kb, illustrate and correctly modify;
Figure 14 ~ 26: the Sequencing chromatogram of wild-type, sample 1 ~ 10,13,15;
Figure 27: sgRNA expresses framework mode figure; This carrier is the basic framework needed for sgRNA expresses;
Figure 28: the Cas9 expression vector mode chart of tissue specific expression; This carrier is with Rosa26 expression vector for framework, is made up of 5 ', the 3 ' homology arm of Rosa26, the promotor of tissue specific expression, neo selection markers, LoxP site, Cas9 protein expression sequence, DTA termination area; This carrier, could the expression of activated carrier only in specific tissue.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further described.
Embodiment 1:
1, experiment material
1.
hD Clinging Kit (639648): buy in Clontech company;
2. Premix Taq (D331A), Primerstar (DRO44A), dNTP (4030Q): buy in precious biotechnology (Dalian) company limited (Takara);
3. restriction endonuclease BbsI (R0539L), AsisI (R0630L), HpaI (R0105S): buy in Bo Maisi bio tech ltd, Beijing (NEB); Church Cloning Vector is commercialization plasmid, by Addgene, buys in Church laboratory; PCR-Blunt II-TOPO is also commercialization plasmid, by Addgene, buys in Invitrogen company; Plasmid with Cre recombinase: by Addgene, buys in Albee Messing laboratory; Rosa26 expression vector is originated, and by Addgene, buys in Liqun Luo laboratory.
2, gene structure and sequential analysis
Goal gene: mouse eukaryotic translation initiation factor 3 (Eif3h gene);
Ensembl genes encoding number: ENSMUSG00000022312;
Eif3h gene structure: Eif3h gene contains 8 exons, comprises with the initial exon 1 of ATG with the exon 8 of TAA termination;
Gene describes: eukaryotic translation initiation factor 3 (Eukaryotic translation initiation factors3, Eif3) be the factor maximum and the most complicated in eukaryotic translation initiation factor, playing a significant role in protein transcription initiating process, is the center protein factor connected each other with other translation initiation factors.Eif3 is subunit complex more than, and Mammals comprises at least 12 subunits, and Eif3h is one of them, all high expression level in kinds of tumors tissue, shows that the generation of itself and kinds of tumors, development, malignant behaviors are closely related.
3, find gene knockout site and screen
(1) on Ensembl and NCBI, find Eif3h gene-correlation information, determine to knock out region;
(2) knocking out after region is selected utilizes Crispr software (http://crispr.mit.edu/) to design sgRNA fragment;
(3) on ncbi database, the target spot that Crispr selects is analyzed, select less sgRNA site, site of missing the target, finally target spot is positioned on exon 4.
4, the structure of sgRNA expression vector and mouse make
(1) sgRNA fragment design:
Upstream primer: 5 ’ – AGTTGCTTCAGCGAGAGAGATCCTT – 3 '
Downstream primer: 5 ’ – AAACAAGGATCTCTCTCGCTGAAGC – 3 '
(2) the sgRNA fragment of synthesis is annealed into double-stranded segment; Wherein the condition of this annealing and system as follows:
Upstream primer (100 μMs) 1 μ l
Downstream primer (100 μMs) 1 μ l
10*T4 connects buffer (NEB) 1 μ l
ddH2O 6.5μl
T4 PNK(NEB) 0.5μl
Response procedures: 37 DEG C of 30min
95 DEG C of 5min, are slow cooling to 25 DEG C subsequently, then react 5min.
(3) be connected to by double-stranded segment in the linearized vector cut with BbsI enzyme and obtain connecting product, it is the Church Cloning Vector containing Alb promotor (liver specific expression), and collection of illustrative plates as shown in Figure 5.
(4) by connection product conversion, select positive colony, carry out bacterium colony PCR checking;
Bacterium colony PCR flow process is as follows:
Primer is:
Product primer F:TGTACAAAAAAGCAGGCTTTAAAG
Product primer R:AAACAAGGATCTCTCTCGCTGAAGC
Reaction system:
Response procedures:
(5) product obtained is carried out agarose gel electrophoresis detection, obtain the fragment of one section of 338bp, as shown in Figure 6.
(6) the correct clone of checking is checked order, to guarantee the exactness of sgRNA sequence.
(7) sgRNA expression plasmid correct for order-checking electricity is gone in the ES cell of mouse; Verify through Southern Blot, result as shown in Figure 7 and Figure 8;
(8) will verify that correct ES cell infusion is in the blastaea of mouse, be transferred to subsequently in the female mouse of 2 replace-conceives, finally obtain the mouse that 9 have sgRNA.
The structure of embodiment 2 Cas9 expression vector and the making of Cas9 instrument mouse
(1) amplification of Cas9 albumen
1. the primer of design amplification Cas9 albumen, as follows:
Cas9-F：CGCGGTCTTTCCAGTGATCGATTAGTTATTAATAGTAATCAA
Cas9-R：CTCTAGTCCGCGGGTGCGATAGCTCACACCTTCCTCTTCTTCTTG
2. pcr amplification Cas9 albumen complete sequence, system is as follows:
PCR program is as follows:
3. PCR primer is carried out agarose gel electrophoresis, as shown in Figure 9.
(2) Cas9 albumen is connected with Rosa26 carrier
1. Clontech company is used
the correct Cas9 sequence of detection is connected with through the linearizing carrier of AsisI and HpaI enzyme by HD Clinging Kit test kit.Carrier is corporate makeover, its promotor CMV, the 5 ' arm of Rosa26, Rosa26 carrier framework of 3 ' arm of expressing with broad spectrum, and collection of illustrative plates as shown in Figure 10.
2., after the vector intestinal bacteria connected, picked clones, bacterium colony PCR being verified, Suzhou Jin Weizhi company is sent to check order.Bacterium colony PCR primer is as follows:
Cas9-SF：CACCATAGACAGAAAGCGGTACACC
Cas9-SR：CTAAAGCGCATGCTCCAGACTG
Reaction system is as follows:
Response procedures is as follows:
(3) electricity after vector linearization correct for order-checking is forwarded in ES cell;
(4) positive ES cells containing Cas9 albumen is gone out with the Screening of Media containing Liu Suanyan NEOMYCIN SULPHATE; Subsequently with SouthernBlot checking, result as shown in Figures 12 and 13.
(5) plasmid with Cre recombinase is forwarded in positive ES cells, obtain the positive ES cells not having selection markers;
(6) will verify that correct ES cell microinjection is in Mouse Blastocysts, be then transferred in the female mouse of 2 replace-conceives, finally obtain 10 instrument mouse containing Cas9 albumen.
The acquisition of embodiment 3 conditionality knock-out mice
Cas9 instrument mouse and sgRNA mouse are hybridized, final acquisition 15 mouse, get the different tissues of mouse, extract genome, after carrying out pcr amplification with Eif3h-F/Eif3h-R primer pair, be sent to the order-checking of Suzhou Jin Weizhi company, in result display liver organization, gene knockout rate reaches more than 80%, and the gene of its hetero-organization is then intact.
Above-mentioned primer pair: Eif3h-F:ATCATATATTTAATTTTCAACAAGT
Eif3h-R：CTTTCCTACAGAGCTTCACCT
Gene knockout interpretation of result:
Liver organization:
Wild-type refers to the liver organization of the mouse not doing any modification;
1 ~ No. 15 sample refers to the different tissues getting 15 mouse respectively and tests, extract the genome of different tissues respectively, carry out pcr amplification, send to order-checking subsequently, result display only has the Eif3h gene of liver organization then intact by this gene modifying its hetero-organization; 11, the Eif3h gene of the liver organization of 12, No. 14 samples is not modified, identical with wild-type.
After order-checking, the main peak of wild-type is:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
No. 1 sample:
Main peak:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak:
ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC
(deleting 1bp)
No. 2 samples
Main peak:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak:
ATCCCATAAAAACTGCCCAA-CTTCTCTCTCGCTGAAGGCGTACAGACTGAC
(deleting 1bp)
No. 3 samples
Main peak:
ATCCCATAAAAACTGCCCAAG-GATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak:
ATCCCATAAAAACTGCCCAAGAGATCTCTCTCGCTGAAGGCGTACAGACTGAC (inserting 1bp)
No. 4 samples
Main peak:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak:
ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC (deleting 1bp)
No. 5 samples
Main peak: ATCCCATAAAAACTGCCCAAGG---TCTCTCGCTGAAGGCGTACAGACTGAC (deleting 3bp)
Secondary peak: ATCCCATAAAAACTGC-----------------TGAAGGCGTACAGACTGAC (deleting 17bp)
No. 6 samples
Main peak:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak:
ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC (deleting 1bp)
No. 7 samples
Main peak:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak:
ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC
(deleting 1bp)
No. 8 samples
Main peak:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak: ATCCCATAAAAACTGCCCAA---TCTCTCTCGCTGAAGGCGTACAGACTGAC (deleting 3bp)
No. 9 samples
Main peak:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak: ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC (deleting 1bp)
No. 10 samples
Main peak:
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak: ATCCCATAAAAACTGCCCAA-CTTCTCTCTCGCTGAAGGCGTACAGACTGAC (deleting 1bp)
No. 13 samples
Main peak:
ATCCCATAAAAACTGCCCAAG-GATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak:
ATCCCATAAAAACTGCCCAAGAGATCTCTCTCGCTGAAGGCGTACAGACTGAC (inserting 1bp)
No. 15 samples
Main peak:
ATCCCATAAAAACTGCCCAAG-GATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak:
ATCCCATAAAAACTGCCCAAGAGATCTCTCTCGCTGAAGGCGTACAGACTGAC (inserting 1bp)
The sequencing result of above-mentioned sample 1 ~ 10,13,15 correspondence is see shown in Figure 15 ~ 26.
The structure of the sgRNA carrier of embodiment 4 constitutive expression, the Cas9 carrier of tissue specific expression
SgRNA expression vector is pCR-Blunt II-TOPO carrier, as shown in figure 27;
Cas9 albumen for prototype with Rosa26 carrier, is inserted into mouse Rosa26 region, avoids the cytotoxicity that the radom insertion of Cas9 albumen causes, as shown in figure 28 by the Cas9 carrier of tissue specific expression;
Other structure flow process is with embodiment 1 and embodiment 2, result is with the result part of embodiment 3, only have the Eif3h gene in liver organization to be modified, this gene in its hetero-organization is then intact, and this illustrates that this scheme can reach the object of conditional gene knockout equally.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the primer for gene knockout, it is characterized in that, comprise following five groups of primers, first group of primer sequence is as shown in SEQ NO:1 and SEQ NO:2, second group of primer sequence is as shown in SEQ NO:3 and SEQ NO:4,3rd group of primer sequence is as shown in SEQ NO:5 and SEQ NO:6, and the 4th group of primer sequence is as shown in SEQNO:7 and SEQ NO:8, and the 5th group of primer sequence is as shown in SEQ NO:9 and SEQ NO:10.
2. the application of primer according to claim 1 in gene knockout.
3., based on a conditional gene knockout method for CRISPR/Cas9 technology, it is characterized in that, comprise the following steps:
1) structure of expression vector: build the sgRNA carrier of tissue specific expression and the Cas9 carrier of composing type/drug induced expression;
2), after expression vector order-checking correctly, electricity forwards in ES cell respectively; Verified by PCR or Southern Blot, obtain ES cell containing sgRNA expression plasmid respectively and containing the ES cell of Cas9 albumen and enlarged culturing;
3) by the Plastid transformation containing Cre recombinase in the ES cell containing Cas9 albumen, obtain and there is no the ES cell of the Cas9 expression vector of selection markers;
4) the ES cell containing Cas9 expression vector and the ES cell containing sgRNA expression plasmid are expelled in blastaea respectively;
5) blastaea is transplanted to respectively in the female mouse of replace-conceive, raises; The offspring produced is F
0for the Cas9 instrument mouse of the sgRNA mouse of tissue specific expression and composing type/drug induced;
6) by the correct Cas9 instrument mouse of detection and sgRNA mouse hybrid, tissue specificity/drug induced knock out mice is obtained.
4. gene knockout method according to claim 3, is characterized in that, the sgRNA carrier of described tissue specific expression comprises the promotor of tissue specific expression, Cre recombinase, reverse 2 LoxP sites and reverse U6 promotor.
5. gene knockout method according to claim 3, it is characterized in that, the sgRNA carrier of described tissue specific expression comprises 2 identical LoxP sites of the promotor of tissue specific expression, Cre recombinase, the U6 promotor of forward, direction and termination area.
6. gene knockout method according to claim 3, is characterized in that, described F
0construction process for tissue specific expression sgRNA mouse is as follows:
21) sgRNA fragment Design and synthesis:
22) by step 21) the sgRNA fragment of synthesizing is annealed into double-stranded segment;
23) double-stranded segment is connected in linearized vector Church Cloning Vector or pCR-Blunt II-TOPO and obtains connecting product sgRNA expression plasmid;
24) connection product sgRNA expression plasmid is transformed, selects positive colony, carry out bacterium colony PCR checking;
25) product that bacterium colony PCR obtains is carried out agarose gel electrophoresis detection;
26) obtain verifying that correct clone carries out checking order the correct sgRNA expression plasmid that checks order;
27) sgRNA expression plasmid correct for order-checking electricity is gone in the ES cell of mouse; Correct ES cell is verified through Southern Blot checking;
28) will verify that correct ES cell infusion is in the blastaea of mouse, be transferred to subsequently in the female mouse of replace-conceive, finally obtain F
0for the mouse of sgRNA.
7. gene knockout method according to claim 3, is characterized in that, the construction process of the Cas9 instrument mouse of described composing type/drug induced is as follows:
31) amplification of Cas9 albumen;
32) Cas9 albumen is connected the Cas9 carrier obtaining composing type/drug induced expression with Rosa26 carrier;
33) electricity after the Cas9 vector linearization of composing type/drug induced expression correct for order-checking is forwarded in ES cell to obtain containing Cas9 albumen ES cell;
34) positive ES cells containing Cas9 albumen is obtained with the ES cell that the Screening of Media containing Liu Suanyan NEOMYCIN SULPHATE contains Cas9 albumen; Subsequently with Southern Blot checking;
35) plasmid with Cre recombinase is forwarded to containing in Cas9 protein positive ES cell, obtain the positive ES cells not having selection markers;
36) will the positive ES cells microinjection of selection markers be there is no in Mouse Blastocysts, be then transferred in the female mouse of replace-conceive, finally obtain the instrument mouse containing Cas9 albumen.
8. gene knockout method according to claim 6, is characterized in that, the primer sequence that described bacterium colony PCR adopts is as shown in SEQ NO:3 and SEQ NO:4, and the amplification condition of bacterium colony PCR is: denaturation 94 DEG C, 3min; Sex change 94 DEG C, 20s; Anneal 58 DEG C, 25s; Extend 72 DEG C, 20s, 25 circulations, extend 72 DEG C eventually, 5min.
9. gene knockout method according to claim 9, is characterized in that, the amplification step of described Cas9 albumen is as follows: the primer of design amplification Cas9 albumen, and its sequence is as shown in SEQ NO:5 and SEQ NO:6; Adopt pcr amplification Cas9 albumen complete sequence, pcr amplification condition is: denaturation 98 DEG C, 3min; Sex change 98 DEG C, 25s; Anneal 63 DEG C, 25s; Extend 72 DEG C, 4min30s, 25 circulations, extend 72 DEG C eventually, 5min.
10. gene knockout method according to claim 7, is characterized in that, described Cas9 albumen and Rosa26 carrier Connection Step as follows: with Clontech company
the correct Cas9 sequence of detection is connected with through the linearizing carrier of AsisI and HpaI enzyme by HD Clinging Kit test kit, after the vector intestinal bacteria connected, picked clones, bacterium colony PCR are verified, Suzhou Jin Weizhi company is sent to check order, what wherein bacterium colony PCR primer adopted is the primer shown in SEQNO:7 and SEQ NO:8, the amplification condition of this bacterium colony PCR is: denaturation 95 DEG C, 3min; Sex change 95 DEG C, 30s; Anneal 58 DEG C, 30s; Extend 72 DEG C, 45s, 26 circulations, extend 72 DEG C eventually, 5min.
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