CN104404036B - Conditional gene knockout method based on CRISPR/Cas9 technologies - Google Patents
Conditional gene knockout method based on CRISPR/Cas9 technologies Download PDFInfo
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Abstract
The invention discloses a kind of primer for gene knockout, including five groups of primers, respectively such as SEQ NO:1 and SEQ NO:2、SEQ NO:3 and SEQ NO:4、SEQ NO:5 and SEQ NO:6、SEQ NO:7 and SEQ NO:8、SEQ NO:9 and SEQ NO:Shown in 10.The invention also discloses application of the described primer in terms of gene knockout.The invention also discloses the conditional gene knockout method based on CRISPR/Cas9 technologies.The present invention can carry out condition specificity, Space-time speciality, drug induced genetic modification；The harm to other cells is reduced, studies function of the gene of constitutive expression in particular organization；Only need Cas9 instruments mouse that tissue, Region-specificity gene knockout or inducible genes can be achieved to knock out；Test period is short, saves time and cost.
Description
Technical field
The invention belongs to genetic modification technical field, and in particular to the conditional gene based on CRISPR/Cas9 technologies strikes
Except method.
Background technology
With the progress of science and technology and the continuous exploration to life science, people are in vivo a certain gene in specific group
Knit, the research of cell and the expression in the time seems more urgent.The locus specificity restructuring skill developed rapidly in recent years
Art is to adapt to this needs and caused key gene operation instrument, and it can be in the duration of certain stages or in specifically organizing
Target gene inactivation is induced, embryo's Deaths caused by target gene missing is avoided or complex phenotypes occurs, delete selection markers
Gene, so as to make complete functional analysis to target gene.The restructuring enzyme system being widely used at present has Cre-LoxP systems
System, FLP-FRT systems, R-RS systems and I-SceI systems, but the still Cre-LoxP systems that application is most.
Cre-LoxP restructuring enzyme system is that conditional gene is practiced shooting, induced gene is practiced shooting, Space-time speciality gene targeting
The core technology of strategy.
Cre-LoxP systemic origins include following two compositions in P1 bacteriophages：
1. one section of 34bp DNA sequence dna, the core sequence of inverted repeats and a 8bp containing two 13bp.This
Section 34bp sequences are the recognition sites of recombinase, are referred to as LoxP sites.
2. Cre recombinases, it is a kind of monomeric protein being made up of 343 amino acid, can trigger the DNA in LoxP sites
Restructuring.The DNA of any sequence, when it is between two LoxP sites, otherwise lacked in the presence of Cre recombinases
Lose (direction in two LoxP sites is identical), otherwise direction occurs to reverse (two LoxP sites in opposite direction), such as Fig. 1 institutes
Show.
Realize that the knockout of certain specific gene under given conditions is, it is necessary to which two transgenosis are small in vivo using Cre-LoxP systems
Mouse.First mouse：Build a target gene in vitro first, a LoxP site is contained at its both ends respectively, afterwards by structure
This section of gene order built up is transferred in embryonic stem cell.Treated embryonic stem cell is implanted to the uterus of pseudopregnant mouse
It is interior, it is budded into a complete embryo again, eventually become a transgenic mice.In this transgenic mice,
LoxP sites are introduced in the introne of corresponding gene, and the function of corresponding gene will not be had an impact in theory, therefore one
As in the case of, the phenotype of the mouse is normal.Second transgenic mice：Typically done using egg mother cell injection or embryo
Cell technology is obtained, and in this mouse, Cre recombinases are placed under the regulation and control of certain specific gene promoter, can make it
Expressed under the conditions of certain is specific.Finally, allow this two mouse mating, the filial generation of above two genotype is contained while generation
Mouse will lack a certain specific gene in a certain certain types of cell.
Artificial endonucleases Clustered Regularly Interspaced Short Palindromic
Repeats, CRISPRs/Cas9 system, it is widely present in prokaryotes (most bacterium and all archeobacterias) genome
In.CRISPRs/Cas9 systems have been caused with its unique structure with special function always since 2002 are defined first
The common concern of scientists from all over the world.CRISPR is roughly divided into 3 classes, and what it is for genetic modification is II type CRISPR systems, its composition
It is relatively simple, it is only necessary to the RNA of Cas9 and two non-coding：CrRNA and trans-activation crRNA (tracrRNA), three components
The targeting of exogenous dna fragment can be mediated to degrade.In the operating process of reality, we only need to design specific sgRNA,
And it is transferred to together with Cas9 albumen in the embryonated egg of mouse.
Although the conditionity that Cre-loxp restructuring enzyme system is capable of mediated gene knocks out, avoid caused by lacking functional gene
The inferior position of traditional gene targeting such as embryo's premature death, but exist many uncertain：
1. implicit/false LoxP sequences in mammalian genome be present, its sequence and LoxP conserved sequence may
It is not fully identical, but can be identified and recombinate by Cre recombinases, and then cause unnecessary DNA damage.
2. poor specificity, hardly result in the progeny mice that double transgenic be present, success rate<50%.
3. evaluation and screening process is complicated, time-consuming.
Existing CRISPR/Cas9 systems are although simple to operate, and targeting accuracy is high, but can not realize tissue, cell, when
Empty specific knockdown.
The content of the invention
Goal of the invention：First technical problem to be solved by this invention there is provided a kind of drawing for gene knockout
Thing.
Second technical problem to be solved by this invention there is provided application of the above-mentioned primer in terms of gene knockout.
3rd technical problem to be solved by this invention there is provided a kind of conditionity based on CRISPR/Cas9 technologies
Gene knockout method.
The tissue specificity of this research knocks out technology, is by the third generation genetic modification technology CRISPR/Cas9 and Cre-
LoxP system globe areas.The advantages of its existing CRISPR/Cas9 technology, and can realize that tissue specificity/medicine of gene lures
Conductivity type gene knockout；Other, efficiency high shorter than the cycle of Cre-LoxP technology again, decreases cytotoxicity.The present invention only needs
Design specific sgRNA, you can realize tissue, the special sex modification of space-time of gene.Can using Cas9 instrument mouse technologies
With function of the gene for studying constitutive expression in a short time efficiently, special in a certain tissue or specifically time, it is
The research of human diseases provides theoretical foundation and animal model.This other invention also can be as clinical and medical research supporting skill
Art, escort for the health of the mankind.
Technical scheme：In order to solve the above problems, the technical scheme is that providing one group is used for gene knockout
Primer, including following five groups of primers, first group of primer sequence such as SEQ NO:1 and SEQ NO:Shown in 2, second group of primer sequence is such as
SEQ NO:3 and SEQ NO:Shown in 4, the 3rd group of primer sequence such as SEQ NO:5 and SEQ NO:Shown in 6, the 4th group of primer sequence
Such as SEQ NO:7 and SEQ NO:Shown in 8, the 5th group of primer sequence such as SEQ NO:9 and SEQ NO:Shown in 10.
Application of the above-mentioned primer in terms of gene knockout.
A kind of conditional gene knockout method based on CRISPR/Cas9 technologies, comprises the following steps：
1) structure of expression vector：Build sgRNA carriers and the composing type/drug induced expression of tissue specific expression
Cas9 carriers；
2) after expression vector sequencing is correct, electricity is gone in ES cells respectively；Verified by PCR or Southern Blot, point
The ES cells containing sgRNA expression plasmids and the ES cells containing Cas9 albumen are not obtained and expand culture；
3) plasmid containing Cre recombinases is transformed into the ES cells containing Cas9 albumen, obtains no selection markers
Cas9 expression vectors ES cells；
4) the ES cells containing Cas9 expression vectors and the ES cells containing sgRNA expression plasmids are expelled to blastaea respectively
In；
5) blastaea is transplanted in replace-conceive dams respectively, raised；The offspring produced is F0For tissue specificity
The Cas9 instrument mouse of the sgRNA mouse of expression and composing type/drug induced；
6) correct Cas9 instruments mouse and sgRNA mouse hybrids will be detected, obtains tissue specificity/drug induced gene
Knock-out mice.
Wherein, the sgRNA carriers of above-mentioned tissue specific expression include the promoter of tissue specific expression, Cre restructuring
Enzyme, 2 reverse LoxP sites and reverse U6 promoters, it is set to express Cre enzymes, guiding LoxP positions in particular organization
U6 promoter inversions between point, so as to start sgRNA expression, as shown in Figure 2.
Wherein, the sgRNA carriers of above-mentioned tissue specific expression include the promoter of tissue specific expression, Cre restructuring
Enzyme, positive U6 promoters, 2 LoxP sites of direction identical and termination area, make it only express Cre in particular organization
Enzyme, the termination area between two LoxP sites is knocked out, so as to start sgRNA expression framework.As shown in Figure 3.
Wherein, above-mentioned F0It is as follows for the construction method of tissue specific expression sgRNA mouse：
21) design of sgRNA fragments and synthesis：The sgRNA fragment sequences such as SEQ NO:1 and SEQ NO:Shown in 2；It is described
SgRNA fragment specific designs are as follows：
(a) relevant information of target gene is found on Ensembl and NCBI, it is determined that knocking out region；
(b) knock out after region is selected and utilize Crispr softwares (http://crispr.mit.edu/) design sgRNA pieces
Section；
(c) target spot of Crispr selections is analyzed on ncbi database, selects the less sgRNA positions in site of missing the target
Point；
22) the sgRNA fragments of synthesis are annealed into double-stranded segment；
23) double-stranded segment is connected to linearized vector Church Cloning Vector or pCR-Blunt II-TOPO
In obtain connection product sgRNA expression plasmids；
24) connection product sgRNA expression plasmids converted, select positive colony, carry out bacterium colony PCR checkings；
25) the obtained products of bacterium colony PCR are entered into row agarose gel electrophoresis detection；
26) checking correctly is cloned to be sequenced to obtain and correct sgRNA expression plasmids is sequenced；
27) correct sgRNA expression plasmids electricity will be sequenced to go in the ES cells of mouse；Verified through Southern Blot；
28) it will verify that correct ES cell infusions into the blastaea of mouse, are subsequently transferred in replace-conceive dams, finally obtained
F0For sgRNA mouse.
Wherein, the construction method 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 to obtain the Cas9 carriers of composing type/drug induced expression with Rosa26 carriers；
33) electricity is gone in ES cells after the Cas9 vector linearizations of correct composing type/drug induced expression being sequenced；
34) contained with the Cas9 carriers of the Screening of Media composing type containing neomycin/drug induced expression
The positive ES cells of Cas9 albumen；Then verified with Southern Blot；
35) plasmid with Cre recombinases is gone in the positive ES cells containing Cas9 albumen, obtained without screening mark
The positive ES cells of note；
36) by the positive ES cells microinjection of no selection markers into Mouse Blastocysts, replace-conceive dams are then transferred to
In, finally obtain the instrument mouse containing Cas9 albumen.
Wherein, the primer sequence such as SEQ NO that above-mentioned bacterium colony PCR is used:3 and SEQ NO:Shown in 4, bacterium colony PCR amplification
Condition is：94 DEG C of pre-degeneration, 3min；94 DEG C of denaturation, 20s；58 DEG C of annealing, 25s；72 DEG C, 20s, 25 circulations of extension, eventually extension
72 DEG C, 5min.
Wherein, the amplification step of above-mentioned Cas9 albumen is as follows：The primer of design amplification Cas9 albumen, its sequence such as SEQ
NO:5 and SEQ NO:Shown in 6；Cas9 albumen complete sequences are expanded using PCR, PCR amplification conditions are：98 DEG C of pre-degeneration, 3min；Become
98 DEG C of property, 25s；63 DEG C of annealing, 25s；72 DEG C, 4min30s, 25 circulations of extension, eventually 72 DEG C of extension, 5min.
Wherein, above-mentioned Cas9 albumen and Rosa26 carrier Connection Steps are as follows：With Clontech companiesHD
Clinging Kit kits will detect correct Cas9 sequences and are connected with through AsisI with the carrier that HpaI enzymes linearize, by even
After the carrier connected converts Escherichia coli, picked clones, bacterium colony PCR checkings, Suzhou Jin Weizhi companies are sent to be sequenced, wherein bacterium colony
PCR primer is using SEQ NO:7 and SEQ NO:Primer shown in 8, bacterium colony PCR amplification condition are：95 DEG C of pre-degeneration,
3min；95 DEG C of denaturation, 30s；58 DEG C of annealing, 30s；72 DEG C, 45s, 26 circulations of extension, eventually 72 DEG C of extension, 5min.
Cas9 albumen is inserted into the Rosa26 regions of mouse, avoids Cas9 albumen by we using Rosa26 carriers as prototype
Radom insertion caused by cytotoxicity, as shown in Figure 4.
Beneficial effect：The present invention has advantages below relative to prior art：Condition based on CRISPR/Cas9 technologies
The advantages of property gene Knockout：
1. simple to operate, high efficiency, low fatal rate, limited without species.
2. condition specificity, Space-time speciality, drug induced genetic modification can be carried out.
3. reducing the harm to other cells, function of the gene of constitutive expression in particular organization is studied；Avoid work(
Can embryo's premature death caused by the missing of gene.
4. only need a Cas9 instruments mouse that tissue, Region-specificity gene knockout or inducible genes can be achieved to knock out.
5. the test period is short, time and cost are saved.
Brief description of the drawings
Fig. 1：The conditional gene knockout ideograph of Cre-LoxP recombinase System-mediateds；The DNA of any sequence, when its position
When between two LoxP sites, if LoxP sites direction is identical, sequence deletion；If LoxP sites are in opposite direction, sequence
Reverse；
Fig. 2：The sgRNA expression vector ideographs of tissue specific expression；The carrier be by tissue-specific promoter,
Cre recombinases, reverse LoxP sites, reverse U6 promoters and sgRNA expression framework composition；The carrier is only specific
Tissue in, can just excite the expression of Cre recombinases, and then mediate U6 promoters forward direction between two reverse LoxP sites simultaneously
Start the expression of sgRNA expression frameworks；
Fig. 3：The sgRNA expression vector ideographs of tissue specific expression；The carrier be by tissue-specific promoter,
Cre recombinases, U6 promoters, LoxP sites, terminator and sgRNA expression framework compositions；The carrier is only specifically being organized
In, the expression of Cre recombinases can be just excited, and then the termination area between two LoxP sites is knocked out, so as to start sgRNA expression
The expression of framework；
Fig. 4：The Cas9 expression vector ideographs of broad spectrum activity expression；The carrier be using Rosa26 expression vectors as framework, by
Rosa26 5 ', 3 ' homology arms, composing type/drug induced promoter, neo selection markers, LoxP sites, Cas9 protein expressions
Sequence, DTA termination area composition；Transfer them in Mice Body, mouse will be inserted into using homologous recombination technique
Rosa26 regions；
Fig. 5：The sgRNA expression vectors of liver specific expression；The carrier is the Alb startups by liver specific expression
Son, Cre recombinases, reverse LoxP sites, reverse U6 promoters and sgRNA expression framework composition；The carrier only exists
In liver, the expression of Cre recombinases can be just excited, and then mediates the U6 promoters between two reverse LoxP sites positive and opens
The expression of dynamic sgRNA expression framework；
Fig. 6：The bacterium colony PCR proof diagrams that sgRNA is connected with carrier；Obtained primer size is 338bp.DNA Marker bars
Band size from bottom to top be respectively 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp,
1000bp；
Fig. 7~8：SgRNA expression plasmid electricity is gone in the ES cells of mouse, with Southern Blot the result figures；Base
Because group is after Xba I digestions, the figure after being hybridized with Alb probes, Cre and U6 probes, illustrate that sgRNA has completely been transferred to ES
In cell；
Fig. 9：Cas9 albumen amplification in vitro electrophoretograms；3.9kb fragment is obtained after PCR amplifications；DNA Marker bands are big
It is small from bottom to top be respectively 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp, 2500bp, 3000bp, 3500bp,
4000bp、5000bp、6000bp、8000bp、10000bp；
Figure 10：The Cas9 protein expression vector ideographs of broad spectrum activity expression；The carrier is using Rosa26 expression vectors as frame
Frame, by the 5 ' of Rosa26,3 ' homology arms, CMV strong promoters, neo selection markers, LoxP sites, Cas9 protein expressions sequence,
DTA termination area forms；Transfer them in Mice Body, homologous recombination technique will be utilized to be inserted into the Rosa26 areas of mouse
Domain；
Figure 11：The bacterium colony PCR proof diagrams that sgRNA is connected with carrier；Obtained primer size is 601bp；DNA Marker
Stripe size from bottom to top be respectively 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp,
1000bp；
Figure 12~13：Cas9 expression plasmid electricity is gone in the ES cells of mouse, with Southern Blot the result figures；
3 ' end checkings：After Hind II digestions, wild-type fragment 9.3kb, saltant type 7.6kb；5 ' end checkings：After EcoR V digestions,
Wild-type fragment 11.6kb, saltant type 5.2kb, illustrate correct modification；
Figure 14~26：The Sequencing chromatogram of wild type, sample 1~10,13,15；
Figure 27：SgRNA expresses framework ideograph；The carrier is the basic framework needed for sgRNA expression；
Figure 28：The Cas9 expression vector ideographs of tissue specific expression；The carrier is using Rosa26 expression vectors as frame
Frame, by the 5 ' of Rosa26,3 ' homology arms, the promoter of tissue specific expression, neo selection markers, LoxP sites, Cas9 albumen
Expressed sequence, DTA termination area composition；The carrier is only in specific tissue, the expression of ability activated carrier.
Embodiment
The present invention is further described below in conjunction with the accompanying drawings.
Embodiment 1：
1st, experiment material
①HD Clinging Kit(639648)：Buy in Clontech companies；
②Premix Taq(D331A)、Primerstar(DRO44A)、dNTP(4030Q)：Buy in precious bioengineering
(Dalian) Co., Ltd (Takara)；
3. restriction endonuclease BbsI (R0539L), AsisI (R0630L), HpaI (R0105S)：Buy in Beijing
Bo Maisi bio tech ltd (NEB)；Church Cloning Vector are commercialization plasmids, pass through Addgene, purchase
Buy in Church laboratories；PCR-Blunt II-TOPO are also commercialization plasmid, by Addgene, are bought in Invitrogen
Company；Plasmid with Cre recombinases：By Addgene, buy in Albee Messing laboratories；Rosa26 expression vectors
Source, by Addgene, buy in Liqun Luo laboratories.
2nd, gene structure and sequence analysis
Target gene：Mouse eukaryotic translation initiation factor 3 (Eif3h genes)；
Ensembl gene codes number：ENSMUSG00000022312；
Eif3h gene structures：Eif3h genes contain 8 extrons, including the exon 1 that is originated with ATG and are terminated with TAA
Exon 8；
Gene describes：(the Eukaryotic translation initiation of eukaryotic translation initiation factor 3
Factors3, Eif3) it is the factor maximum in eukaryotic translation initiation factor and most complicated, in protein transcription initiating process
Play a significant role, be the center protein factor connected each other with other translation initiation factors.Eif3 is one multi-subunit compound
Body, it is one of those that mammal, which includes at least 12 subunits, Eif3h, and all high expression, shows it in kinds of tumors tissue
It is closely related with the generation, development, malignant behaviors of kinds of tumors.
3rd, find gene knockout site and screen
(1) Eif3h gene-correlation information is found on Ensembl and NCBI, it is determined that knocking out region；
(2) knock out after region is selected and utilize Crispr softwares (http://crispr.mit.edu/) design sgRNA pieces
Section；
(3) target spot of Crispr selections is analyzed on ncbi database, selects the less sgRNA positions in site of missing the target
Point, finally target spot is positioned on exon 4.
4th, the structure of sgRNA expression vectors and mouse make
(1) sgRNA fragments design：
Sense primer：5’–AGTTGCTTCAGCGAGAGAGATCCTT–3’
Anti-sense primer：5’–AAACAAGGATCTCTCTCGCTGAAGC–3’
(2) the sgRNA fragments of synthesis are annealed into double-stranded segment；Wherein the condition of the annealing and system are as follows：
The μ l of sense primer (100 μM) 1
The μ l of anti-sense primer (100 μM) 1
10*T4 connections buffer (NEB) 1 μ l
ddH2O 6.5μl
T4 PNK(NEB) 0.5μl
Response procedures：37℃ 30min
95 DEG C of 5min, are then slow cooling to 25 DEG C, then react 5min.
(3) double-stranded segment is connected to and obtains connection product with the linearized vector of BbsI digestions, it is to be opened containing Alb
The Church Cloning Vector of mover (liver specific expression), collection of illustrative plates are as shown in Figure 5.
(4) connection product converted, select positive colony, carry out bacterium colony PCR checkings；
Bacterium colony PCR flows are as follows：
Primer is：
Product primer F:TGTACAAAAAAGCAGGCTTTAAAG
Product primer R:AAACAAGGATCTCTCTCGCTGAAGC
Reaction system：
Response procedures：
(5) obtained product is entered into row agarose gel electrophoresis detection, obtains one section of 338bp fragment, as shown in Figure 6.
(6) by checking, correctly clone is sequenced, to ensure the correctness of sgRNA sequences.
(7) correct sgRNA expression plasmids electricity will be sequenced to go in the ES cells of mouse；Verified through Southern Blot,
As a result as shown in Figure 7 and Figure 8；
(8) it will verify that correct ES cell infusions into the blastaea of mouse, are subsequently transferred in 2 replace-conceive dams, finally
Obtain 9 mouse with sgRNA.
The structure of the Cas9 expression vectors of embodiment 2 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 expands Cas9 albumen complete sequences, system is as follows：
PCR programs are as follows：
3. PCR primer is entered into row agarose gel electrophoresis, as shown in Figure 9.
(2) Cas9 albumen is connected with Rosa26 carriers
1. with Clontech companiesHD Clinging Kit kits will detect correct Cas9 sequences
It is connected with through AsisI with the carrier that HpaI enzymes linearize.Carrier is corporate makeover, its promoter CMV with broad spectrum activity expression,
Rosa26 5 ' arm, 3 ' arm Rosa26 carrier frameworks, collection of illustrative plates are as shown in Figure 10.
2. after the carrier connected is converted into Escherichia coli, picked clones, bacterium colony PCR checkings, send gold only intelligence in Suzhou public
Department's sequencing.Bacterium colony PCR primer is as follows：
Cas9-SF：CACCATAGACAGAAAGCGGTACACC
Cas9-SR：CTAAAGCGCATGCTCCAGACTG
Reaction system is as follows：
Response procedures are as follows：
(3) electricity is gone in ES cells after correct vector linearization will be sequenced；
(4) positive ES cells containing Cas9 albumen are gone out with the Screening of Media containing neomycin；Then use Southern
Blot checkings, as a result as shown in Figures 12 and 13.
(5) plasmid with Cre recombinases is gone in positive ES cells, the positive ES for obtaining no selection markers is thin
Born of the same parents；
(6) it will verify that correct ES cell microinjections into Mouse Blastocysts, are then transferred in 2 replace-conceive dams, most
The 10 instrument mouse for comprising only Cas9 albumen are obtained afterwards.
The acquisition of the conditionity knock-out mice of embodiment 3
Cas9 instruments mouse and sgRNA mouse are hybridized, 15 mouse is finally obtained, takes the different tissues of mouse, carry
Genome is taken, after entering performing PCR amplification with Eif3h-F/Eif3h-R primer pairs, the sequencing of Suzhou Jin Weizhi companies is sent to, as a result shows
Gene knockout rate reaches more than 80% in liver organization, 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 for not doing the mouse of any modification；
1~No. 15 sample refers respectively to take the different tissues of 15 mouse to be tested, and extracts different tissues respectively
Genome, enters performing PCR amplification, then sends to sequencing, and the Eif3h genes that as a result display only has liver organization are modified and other groups
The gene knitted is then intact；11st, the Eif3h genes of the liver organization of 12, No. 14 samples are not modified, identical with wild type.
The main peak of wild type is after sequencing：
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 (insertion 1bp)
No. 4 samples
Main peak：
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak：
ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC (deletes 1bp)
No. 5 samples
Main peak：ATCCCATAAAAACTGCCCAAGG---TCTCTCGCTGAAGGCGTACAGACTGAC (deletes 3bp)
Secondary peak：ATCCCATAAAAACTGC-----------------TGAAGGCGTACAGACTGAC (deletes 17bp)
No. 6 samples
Main peak：
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak：
ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC (deletes 1bp)
No. 7 samples
Main peak：
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak：
ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC
(deleting 1bp)
No. 8 samples
Main peak：
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak：ATCCCATAAAAACTGCCCAA---TCTCTCTCGCTGAAGGCGTACAGACTGAC (deletes 3bp)
No. 9 samples
Main peak：
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak：ATCCCATAAAAACTGCCCAAG-ATCTCTCTCGCTGAAGGCGTACAGACTGAC (deletes 1bp)
No. 10 samples
Main peak：
ATCCCATAAAAACTGCCCAAGGATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak：ATCCCATAAAAACTGCCCAA-CTTCTCTCTCGCTGAAGGCGTACAGACTGAC (deletes 1bp)
No. 13 samples
Main peak：
ATCCCATAAAAACTGCCCAAG-GATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak：
ATCCCATAAAAACTGCCCAAGAGATCTCTCTCGCTGAAGGCGTACAGACTGAC (insertion 1bp)
No. 15 samples
Main peak：
ATCCCATAAAAACTGCCCAAG-GATCTCTCTCGCTGAAGGCGTACAGACTGAC
Secondary peak：
ATCCCATAAAAACTGCCCAAGAGATCTCTCTCGCTGAAGGCGTACAGACTGAC (insertion 1bp)
Sequencing result is referring to shown in Figure 15~26 corresponding to above-mentioned sample 1~10,13,15.
The structure of the sgRNA carriers of the constitutive expression of embodiment 4, the Cas9 carriers of tissue specific expression
SgRNA expression vectors are pCR-Blunt II-TOPO carriers, as shown in figure 27；
The Cas9 carriers of tissue specific expression are inserted into mouse Rosa26 using Rosa26 carriers as prototype, by Cas9 albumen
Region, cytotoxicity caused by the radom insertion of Cas9 albumen is avoided, as shown in figure 28；
Others structure flow is with embodiment 1 and embodiment 2, as a result with the result part of embodiment 3, only liver organization
In Eif3h genes be modified, the gene in its hetero-organization is then intact, and this illustrates that this scheme can equally reach conditionity base
Because of the purpose of knockout.
Described above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (6)
- A kind of 1. conditional gene knockout method based on CRISPR/Cas9 technologies, it is characterised in that comprise the following steps：The structure of expression vector：Build the sgRNA carriers of tissue specific expression and the Cas9 of composing type/drug induced expression Carrier；After expression vector sequencing is correct, electricity is gone in ES cells respectively；Verified by PCR or Southern Blot, obtained respectively ES cells containing sgRNA expression plasmids and the ES cells containing Cas9 albumen simultaneously expand culture；Plasmid containing Cre recombinases is transformed into the ES cells containing Cas9 albumen, obtains the Cas9 of no selection markers The ES cells of expression vector；ES cells containing Cas9 expression vectors and the ES cells containing sgRNA expression plasmids are expelled in blastaea respectively；Blastaea is transplanted in replace-conceive dams respectively, raised；The offspring produced is F0For tissue specific expression The Cas9 instrument mouse of sgRNA mouse and composing type/drug induced；Correct Cas9 instruments mouse and sgRNA mouse hybrids will be detected, it is small to obtain tissue specificity/drug induced gene knockout Mouse；The sgRNA carriers are by tissue-specific promoter, Cre recombinases successively, two reverse LoxP sites, are clipped in two U6 promoters, 20nt target areas, sgRNA frameworks and U6 termination area composition between reverse LoxP sites.
- 2. gene knockout method according to claim 1, it is characterised in that the F0For tissue specific expression sgRNA The construction method of mouse is as follows：21）SgRNA fragments design and synthesis：22）By step 21）The sgRNA fragments of synthesis are annealed into double-stranded segment；23）Double-stranded segment is connected in linearized vector Church Cloning Vector or pCR-Blunt II-TOPO and obtained To connection product sgRNA expression plasmids；24）By the conversion of connection product sgRNA expression plasmids, positive colony is selected, carries out bacterium colony PCR checkings；25）The obtained products of bacterium colony PCR are entered into row agarose gel electrophoresis detection；26）Checking correctly is cloned to be sequenced to obtain correct sgRNA expression plasmids are sequenced；27）Correct sgRNA expression plasmids electricity will be sequenced to go in the ES cells of mouse；Verify to obtain through Southern Blot Verify correct ES cells；28）It will verify that correct ES cell infusions into the blastaea of mouse, are subsequently transferred in replace-conceive dams, finally obtain F0Generation SgRNA mouse.
- 3. gene knockout method according to claim 1, it is characterised in that the Cas9 of the composing type/drug induced The construction method of instrument mouse is as follows：31）The amplification of Cas9 albumen；32）Cas9 albumen is connected to obtain the Cas9 carriers of composing type/drug induced expression with Rosa26 carriers；33）Electricity after the Cas9 vector linearizations of correct composing type/drug induced expression will be sequenced go in ES cells and obtain ES cells containing Cas9 albumen；34）It is thin that the ES cells for containing Cas9 albumen with the Screening of Media containing neomycin obtain the positive ES containing Cas9 albumen Born of the same parents；Then verified with Southern Blot；35）Plasmid with Cre recombinases is gone to containing in Cas9 protein positive ES cells, obtains the sun of no selection markers Property ES cells；36）By the positive ES cells microinjection of no selection markers into Mouse Blastocysts, then it is transferred in replace-conceive dams, most The instrument mouse containing Cas9 albumen is obtained afterwards.
- 4. gene knockout method according to claim 2, it is characterised in that the primer sequence that the bacterium colony PCR is used is such as SEQ NO:3 and SEQ NO:Shown in 4, bacterium colony PCR amplification condition is：94 DEG C of pre-degeneration, 3min；94 DEG C of denaturation, 20s；Annealing 58 DEG C, 25s；72 DEG C, 20s, 25 circulations of extension, eventually 72 DEG C of extension, 5min.
- 5. gene knockout method according to claim 3, it is characterised in that the amplification step of the Cas9 albumen is as follows： The primer of design amplification Cas9 albumen, its sequence such as SEQ NO:5 and SEQ NO:Shown in 6；It is complete using PCR amplification Cas9 albumen Sequence, PCR amplification conditions are：98 DEG C of pre-degeneration, 3min；98 DEG C of denaturation, 25s；63 DEG C of annealing, 25s；72 DEG C of extension, 4min30s, 25 circulations, eventually 72 DEG C of extension, 5min.
- 6. gene knockout method according to claim 3, it is characterised in that the Cas9 albumen is connected with Rosa26 carriers Step is as follows：With the In-Fusion of Clontech companies®HD Clinging Kit kits will detect correct Cas9 sequences It is connected with through AsisI with the carrier that HpaI enzymes linearize, the carrier connected is converted into Escherichia coli, picked clones, bacterium colony After PCR checkings, Suzhou Jin Weizhi companies are sent to be sequenced, wherein bacterium colony PCR primer is using SEQ NO:7 and SEQ NO:Shown in 8 Primer, bacterium colony PCR amplification condition is：95 DEG C of pre-degeneration, 3min；95 DEG C of denaturation, 30s；58 DEG C of annealing, 30s；Extension 72 DEG C, 45s, 26 circulations, 72 DEG C of extension eventually, 5min.
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