CN103820454B - The method of CRISPR-Cas9 specific knockdown people PD1 gene and the sgRNA for selectively targeted PD1 gene - Google Patents

The method of CRISPR-Cas9 specific knockdown people PD1 gene and the sgRNA for selectively targeted PD1 gene Download PDF

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CN103820454B
CN103820454B CN201410077474.6A CN201410077474A CN103820454B CN 103820454 B CN103820454 B CN 103820454B CN 201410077474 A CN201410077474 A CN 201410077474A CN 103820454 B CN103820454 B CN 103820454B
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胡边
黄行许
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Esoteric Biotechnology (Guangzhou) Co., Ltd.
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上海金卫生物技术有限公司
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Abstract

The invention belongs to genetically engineered field, more particularly, relate to the method for CRISPR-Cas9 specific knockdown people PD1 gene and the sgRNA for selectively targeted PD1 gene.The invention provides the method for CRISPR-Cas9 specific knockdown people PD1 gene and the sgRNA for selectively targeted PD1 gene.The sgRNA of the selectively targeted people PD1 gene utilizing the present invention to prepare can accurate targeted human PD1 gene and realize gene knockout.This preparation method's step is simple, sgRNA targeting is good, CRISPR-Cas9 system to knock out efficiency high.

Description

The method of CRISPR-Cas9 specific knockdown people PD1 gene and the sgRNA for selectively targeted PD1 gene
Technical field
The invention belongs to genetically engineered field, more specifically to the method for CRISPR-Cas9 specific knockdown people PD1 gene and the sgRNA for selectively targeted PD1 gene.
Background technology
Rule cluster interval short palindrome duplicated system (clusteredregularlyinterspacedshortpalindromicrepeat; CRISPR-associated, CRISPR-Cas9) be a kind of complex body with endonuclease activity, identify specific DNA sequence dna, carry out specific site cutting and cause double-strand DNA cleavage (Double-strandbreaks, DSB), under the condition not having template, there is non-homogeneous restructuring end and connect (Non-homologousendjoining, NHEJ), cause phase shift mutation (frameshiftmutation), cause gene knockout (Fig. 1).
This technology due to can fast, any gene of target gene group simply, efficiently, thus cause and pay close attention to widely, within 2012, to start picture explode bud out into popularity.Due to its easily operation, can the multiple gene of target simultaneously, can the advantages such as high-throughput preparation, cost be low, Cas9 has become a kind of technology with fastest developing speed (Pennisi, 2013).Just because of its superiority, this technology Nature recommend 2,013 ten to rank first in progress greatly ( http:// www.nature.com/news/365-days-nature-s-10-1.14367), be at the second place in the 2013 ten large progress that Science recommends ( http:// news.sciencemag.org/breakthrough-of-the-year-2013).
Cas9 target cutting DNA is by two kinds of tiny RNA---crRNA(CRISPRRNA) and tracrRNA (trans-activatingcrRNA) and target complement sequence identification principle realize.Two kinds of tiny RNA are fused into a RNA chain now, are called for short sgRNA(singleguideRNA).Therefore, can sgRNA accomplish that specificity, accurately target target gene are that can CRISPR-Cas9 the prerequisite of specific knockdown target gene, no matter misses the target or wrong target, all can affect the specific knockdown of CRISPR-Cas9 to target gene.Therefore, it is possible to the sgRNA designing, prepare accuracy and selectively targeted target gene becomes the gordian technique (Fig. 1) of CRISPR-Cas9 gene knockout.
Immunotherapy of tumors, especially to the blocking-up of immunologic test point, is the current the most successful field of target gene therapy.Immunologic test point refers to and maintains immune self tolerance, and regulates a series of inhibitive ability of immunity reactions of the immune response intensity under physiological condition and time length.Research shows, tumour cell and immunologic test point approach synergy, create the effect of Tumor suppression immunity, particularly the effect of the effector T cell of Tumor suppression antigen-specific.To the blocking-up of immunologic test point, refer to the monoclonal antibody utilizing T cell immunologic test Inhibitory receptor, the combination of specific inhibition Inhibitory receptor and its part, thus the effect of blocking immunity checking mechanism suppressor T cell activation, the antitumous effect of reinforcing effect T cell.
One of immunologic test target spot being used successfully to clinical immunotherapy of tumors is inhibitive ability of immunity acceptor, PD1 gene.The major function of PD1 is the effector T cell activity of restriction peripheral tissues.The PD1 part of tumor cells expression and the PD1 of T cell combine, and suppressor T cell activates associated kinase, thus retarding effect T cell is active.Utilize PD1 monoclonal antibody (MDX-1106, MK3475, CT-011, AMP-224 etc.), suppress PD1 part and PD1 to combine, thus reach the effect for the treatment of tumour.
But, utilize the treatment of PD1 antibody only better in malignant melanoma curative effect, have part curative effect to kidney and lung cancer.Because the treatment utilizing antibody to carry out target gene is also subject to the restriction of some factors: the effect of (1) antibody is the effect of temporary interruption; (2) Inhibitory receptor has multiple, and how to utilize Multiple Antibodies to block multiple Inhibitory receptor does not also have countermeasure simultaneously; (3) be not easy to develop effective antibody; (4) only for extracellular target spot; (5) antibody drug is expensive, etc.
CRISPR-Cas9 is quick, easy, efficient, selectively targeted knocks out gene, knocks out PD1 gene by target, provides a kind of key tactics for realizing immunotherapy of tumors.But can the sgRNA that design, prepare accuracy and selectively targeted PD1 gene become the gordian technique of CRISPR-Cas9 specific knockdown PD1 gene.Object of the present invention will solve this key technical problem exactly, provides corresponding technical scheme, reaches the object of specific knockdown PD1 gene.
Reference:
MaliP,EsveltKM,ChurchGM.Cas9asaversatiletoolforengineeringbiology.Nat.Methods.2013;10(10):957-963.
PennisiE.TheCRISPRcraze.Science,2013;341(6148):833-6.doi:10.1126/science.341.6148.833.
PardollDM.Theblockadeofimmunecheckpointsincancerimmunotherapy.NatureRev.Cancer,2012;12:252-264.
MellmanI,CoukosG,DranoffG.Cancerimmunotherapycomesofage.Nature,2011;480:480-489.
Summary of the invention
Immunodetection blocking treatment tumour Problems existing is carried out: the effect of (1) antibody is the effect of temporary interruption for the existing PD1 of utilization antibody; (2) Inhibitory receptor has multiple, and how utilizing Multiple Antibodies to block multiple Inhibitory receptor does not also have countermeasure; (3) be not easy to develop effective antibody; (4) only for extracellular target spot; (5) develop that antibody drug is time-consuming, effort, expensive, make antibody drug expensive.The present invention designs, has synthesized the sgRNA of one group of selectively targeted PD1 gene in CRISPR-Cas9 specific knockdown people PD1 gene, and respectively this sgRNA and linear pGL3-U6-sgRNA plasmid are connected into carrier, a pair forward can be realized knocking out of PD1 gene with direction sgRNA oligonucleotide carrier Successful transfection cell together with pST1374-NLS-flag-Cas9-ZF plasmid.This application provides and a kind ofly utilize that Cas9/sgRNA is quick, easy, efficient, the strategy of specific knockdown PD1.Efficiently solve and utilize Antybody therapy Problems existing: (1) directly knocks out PD1 gene, permanent effect can be realized; (2) both can knock out for multiple encoding sequences of PD1 simultaneously, also can knock out for multiple target gene simultaneously; (3) efficient sgRNA is provided; (4) both can for outside born of the same parents, also can for target spot in born of the same parents; (5) sgRNA only needs synthetic polyribonucleotides segment in a small amount, just can produce in enormous quantities.
In order to solve the problems of the technologies described above, the technical scheme of the application is as follows:
One, the design of sgRNA oligonucleotide and selection
1. the design of the sgRNA of target PD1 gene:
Because do not use in-vitro transcription, the mode just building general carrier makes.So if no special instructions, the sgRNA sequence in literary composition refers to sgRNA corresponding DNA sequence.
(1) on PD1 gene, select the sequence of 5 '-GGN (19) GG, if do not have the sequence of 5 '-GGN (19) GG, 5 '-GN (20) GG or 5 '-N (21) GG is also passable.
(2) target site of sgRNA on PD1 gene is positioned at the exon of gene.
(3) target site of sgRNA on PD1 gene is positioned on the common exon of different various shear-forms.
(4) in UCSC database, use BLAST with in BLAT or ncbi database, determine that whether the target sequence of sgRNA is unique.
2. the selection of the sgRNA of target PD1 gene:
(1) can not from the initial son of ATG too close to, prevent from transcribing another ATG of downstream after the meeting and start and occur that one by the gene forms of brachymemma, gene complete deactivation can not be ensured;
(2) target site of sgRNA on PD1 gene is positioned at the first half section of whole gene, especially should in the functional domain of gene;
(3) site selecting (10 ~ 30bp) separated by a distance paired.Be conducive to like this forming specific fragment deletion, be also conducive to reduction and miss the target effect;
Two, the double strand oligonucleotide of sgRNA is built
According to the sgRNA selected, 5 ' add that CCGG obtains forward oligonucleotide (Forwardoligo) (as infructescence itself has had 1 or 2 G at 5 ' end, the so omission 1 of correspondence or 2 G) at it; According to the sgRNA selected, obtain the complementary strand of its corresponding DNA, and 5 ' add that AAAC obtains reverse oligonucleotide (Reverseoligo) at it.Synthesize above-mentioned forward oligonucleotide and reverse oligonucleotide respectively, by the paired sex change of forwardoligo and reverseoligo of the sgRNA oligonucleotide of synthesis, annealing, after annealing, form the double-strand that can be connected into U6 carrier for expression of eukaryon, as follows:
Forwardoligo:5′-CCGGNNNNNNNNNNNNNNNNNN
||||||||||||||||||
Reverseoligo:NNNNNNNNNNNNNNNNNNCAAA-5′
Three, the structure of sgRNA oligonucleotide plasmid
1. linearizing pGL3-U6-sgRNA plasmid (structure as shown in Figure 4).
2. the sgRNA double strand oligonucleotide of annealing is connected with linearizing pGL3-U6-sgRNA plasmid and obtains pGL3-U6-hPD1sg plasmid.
3. transform and be coated with Amp+ flat board (50 μ g/ml).
4. with the method qualification positive colony that the universal primer U6 of IDNO.9 checks order.
5.37 DEG C of shaking tables shake bacterium and spend the night and use AxyPrepPlasmidMiniprepKit(AP-MN-P-250) extracting pGL3-U6-hPD1sg plasmid.
Four, transfectional cell obtains PD1 Knockout cells
1, according to Lipofectamine tM2000TransfectionReagent(Invitrogen, operational manual 11668-019), the pST1374-NLS-flag-Cas9-ZF plasmid (structure as shown in Figure 5) being SEQIDNO.11 by the pGL3-U6-hPD1sg plasmid (can be a kind or multiple) respectively with corresponding sgRNA oligonucleotide and sequence mixes, cotransfection cell.
2, detection is cut with T7EN1 enzyme and TA cloning and sequencing confirms that PD1 gene is knocked.
Further, utilize the sgRNA of a pair adjacent (the target initiation site on PD1 gene is at a distance of 5bp-8bp) to significantly improve simultaneously and knock out efficiency.After the sgRNA oligonucleotide design of target PD1, selection and synthesis, the sgRNA oligonucleotide of target PD1 is connected the carrier pGL3-U6-hPD1sg of the sgRNA oligonucleotide obtained containing target PD1 with linearizing pGL3-U6-sgRNA plasmid, obtain in PD1 Knockout cells process at transfectional cell, operate as follows:
1, according to Lipofectamine tM2000TransfectionReagent(Invitrogen; operational manual 11668-019); by two respectively containing the sgRNA oligonucleotide of 1 target PD1 these two carriers of carrier pGL3-U6-hPD1sg(respectively with complementary initiation site on PD1 gene of the sgRNA oligonucleotide of target PD1 at a distance of 5bp-8bp) being SEQIDNO.11 with sequence, pST1374-NLS-flag-Cas9-ZF plasmid mixes, cotransfection cell.
2, detection is cut with T7EN1 enzyme and TA cloning and sequencing confirms that PD1 gene is knocked.
Present invention also offers the sgRNA of selectively targeted PD1 gene, its sequence is as shown in SEQIDNO.24-203.
Carry out immunodetection blocking treatment tumour technology compared to the existing PD1 of utilization antibody, the invention has the advantages that:
(1) effect of antibody is the effect of temporary closure, and the present invention directly knocks out PD1 gene, can realize permanent effect;
(2) Inhibitory receptor has multiple, and how utilizing Multiple Antibodies to close multiple Inhibitory receptor does not also have countermeasure, and the present invention both can knock out for multiple encoding sequences of PD1, also can knock out for multiple target gene;
(3) effective PD1 antibody research and development are very difficult, the invention provides one group of efficient sgRNA for people PD1 gene;
(4) antibody effect can only for extracellular target spot, and the present invention both can for outside born of the same parents, also can for target spot in born of the same parents;
(5) developing antibody drug is time-consuming, effort, an expensive process, makes antibody drug expensive, utilizes sgRNA only to need synthetic polyribonucleotides segment in a small amount, just can produce in enormous quantities.
Accompanying drawing explanation
Fig. 1 Cas9 realizes fixed point cutting and causes DNA double chain-breaking process schematic diagram
CRISPR/Cas9 system oriented identification and shearing thus cause gene knockout to be realized by sgRNA and Cas9.SgRNA determines the targeting of Cas9.
Fig. 2 T7EN1 enzyme cuts the gene people PD1 specificity cutting of qualification sgRNA/Cas9 mediation
With the HEK293T cellular genome extracted for template, use sequence as hPD1testFor and hPD1testRev of SEQIDNO.12 and SEQIDNO.13 for primer carries out pcr amplification, PCR primer is 390bp, purified pcr product.Above-mentioned PCR primer is got 200ng annealing, use T7EN1 enzyme to cut qualification, electrophoresis.As shown in Figure 2, all there is cutting band, and had very high efficiency in the sample added for the sgRNA of people PD1.
The gene locus specific human PD1 that Fig. 3 sgRNA/Cas9 mediates cuts result order-checking
With the cellular genome extracted for template, use sequence as hPD1testFor and hPD1testRev of SEQIDNO.12 and SEQIDNO.13 for primer carries out pcr amplification.Purified pcr product, is connected into TA and clones and send order-checking.Underlined sequences is PAM sequence; (-) expression knocks out.
The structure of Fig. 4 carrier pGL3-U6-sgRNA
The structure of Fig. 5 carrier pST1374-NLS-flag-cas9-ZF
Embodiment
Below in conjunction with accompanying drawing and specific embodiment, technical scheme of the present invention is described further.
For the design of the sgRNA of selectively targeted PD1 gene and synthesis in embodiment 1CRISPR-Cas9 specific knockdown people PD1 gene
1. the design of the sgRNA of targeted human PD1 gene:
(1) on PD1 gene, select the sequence of 5 '-GGN (19) GG, if do not have the sequence of 5 '-GGN (19) GG, 5 '-GN (20) GG or 5 '-N (21) GG is also passable.
(2) target site of sgRNA on PD1 gene is positioned at the exon of gene, so more easily causes the disappearance of fragment or moves frame sudden change, thus reach the object of gene complete deactivation.
(3) target site of sgRNA on PD1 gene is positioned on the common exon of different various shear-forms.
(4) in UCSC database, use BLAST with in BLAT or ncbi database, determine that whether the target sequence of sgRNA is unique, reduce potential site of missing the target.
According to above method, we devise altogether the sgRNA of 180 targeted human PD1 genes, and sequence is respectively as shown in sequence table SEQ IDNO.24-203.
2. the selection of the sgRNA of targeted human PD1 gene:
(1) target sequence of the sgRNA of target PD1 gene on PD1 gene can not from the initial son of ATG too close to, prevent from transcribing another ATG of downstream after the meeting and start and occur that one by the gene forms of brachymemma, gene complete deactivation can not be ensured.
(2) target site of sgRNA on PD1 gene is positioned at the first half section of whole gene, especially should in the functional domain of gene.
(3) site selecting (10 ~ 30bp) separated by a distance paired on PD1 gene.Be conducive to like this forming specific fragment deletion, be also conducive to reduction and miss the target effect.
According to above method, in the sgRNA(sequence comprising PAM sequence of 180 targeted human PD1 genes respectively as shown in sequence table SEQ IDNO.24-203) in the sequence that meets have 12 (respectively as shown in sequence table SEQ IDNO.40,51,52,57,63,78,82,96,101,126,128 or 136), because sequence is more, there is no need to do experimental verification one by one, we therefrom have selected 4 (respectively as sequence table SEQ IDNO.52,57, shown in 78 or 82) carry out subsequent experimental.
3. the synthesis of the sgRNA oligonucleotide of targeted human PD1 gene and structure
According to 4 that select (respectively as shown in sequence table SEQ IDNO.52,57,78 or 82), 5 ' add that CCGG obtains forward oligonucleotide (Forwardoligo) (as infructescence itself has had 1 or 2 G at 5 ' end, so corresponding omission 1 or 2 G) at it; According to the sgRNA selected, obtain its complementary strand, and 5 ' add that AAAC obtains reverse oligonucleotide (Reverseoligo) at it.(synthetic method is see document: Significantimprovementofqualityforlongoligonucleotidesby usingcontrolledporeglasswithlargepores.NucleosidesNucleo tidesNucleicAcids.2005 in synthesis respectively; 24 (5-7): 1037-41.) above-mentioned forward oligonucleotide and reverse oligonucleotide, by the paired sex change of forwardoligo and reverseoligo of the sgRNA oligonucleotide of synthesis, annealing, form the double-strand sgRNA oligonucleotide that can be connected into U6 carrier for expression of eukaryon after annealing, pattern is as follows:
Forwardoligo:5′-CCGGNNNNNNNNNNNNNNNNNN
||||||||||||||||||
Reverseoligo:NNNNNNNNNNNNNNNNNNCAAA-5′
Sex change, annealing system are:
2.5μlforwardOligo(100μM)
2.5μlreverseOligo(100μM)
1μlNEBbuffer2
4 μ l aqua sterilisas
Run according to following touchdown program in PCR instrument: 95 DEG C, 5min; 95 – 85 DEG C at-2 DEG C/s; 85 – 25 DEG C at-0.1 DEG C/s; Holdat4 DEG C.
The 1st sgRNA(selected is as shown in sequence table SEQ IDNO.52), itself forwardoligo and reverseoligo(Forwardoligo and Reverseoligo sequence are respectively as Suo Shi sequence table SEQ IDNO.1 and 2) in pairs sex change, after annealing acquisition can be connected into the double-strand sgRNA oligonucleotide of U6 carrier for expression of eukaryon.
The 2nd sgRNA(selected is as shown in sequence table SEQ IDNO.82), itself forwardoligo and reverseoligo(Forwardoligo and Reverseoligo sequence are respectively as Suo Shi sequence table SEQ IDNO.3 and 4) in pairs sex change, after annealing acquisition can be connected into the double-strand sgRNA oligonucleotide of U6 carrier for expression of eukaryon.
The 3rd sgRNA(selected is as shown in sequence table SEQ IDNO.57), itself forwardoligo and reverseoligo(Forwardoligo and Reverseoligo sequence are respectively as Suo Shi sequence table SEQ IDNO.5 and 6) in pairs sex change, after annealing acquisition can be connected into the double-strand sgRNA oligonucleotide of U6 carrier for expression of eukaryon.
The 4th sgRNA(selected is as shown in sequence table SEQ IDNO.78), itself forwardoligo and reverseoligo(Forwardoligo and Reverseoligo sequence are respectively as Suo Shi sequence table SEQ IDNO.7 and 8) in pairs sex change, after annealing acquisition can be connected into the double-strand sgRNA oligonucleotide of U6 carrier for expression of eukaryon.
Embodiment 2 utilizes CRISPR-Cas9 specific knockdown people PD1 gene (sgRNA for target PD1 gene is as shown in sequence table 52)
1, the pGL3-U6-sgRNA plasmid of linearizing sequence as shown in sequence table SEQ IDNO.10.
Enzyme cuts system and condition is as follows:
2μgpGL3-U6-sgRNA（400ng/μl）；
1μlCutSmartBuffer；
1μlBsaI(NEB，R0535L)；
Moisturizing to 50 μ l, hatches 3 ~ 4 hours for 37 DEG C, vibrates at set intervals and centrifugal in case droplet evaporation covers to pipe.
Enzyme cuts into rear AxyPrepPCRCleanupKit(AP-PCR-250) purifying is recycled in 20 ~ 40 μ l aqua sterilisas.
2, by the double-strand sgRNA oligonucleotide (itself Forwardoligo with Reverseoligo sequence is respectively if sequence table SEQ IDNO.1 is with 2 Suo Shi) that can be connected into U6 carrier for expression of eukaryon of acquisition after sex change, annealing, being connected with linearizing pGL3-U6-sgRNA plasmid obtains pGL3-U6-hPD1sg1 plasmid.
Linked system is as follows:
3 μ l, 50 μMs of annealed product (double-strand sgRNA oligonucleotide, its forwardoligo is as shown in sequence table SEQ IDNO.1, and its reverseoligo is as shown in sequence table SEQ IDNO.2)
The linearizing pGL3-U6-sgRNA plasmid of 1 μ l (25ng/ μ l)
1μlT4ligationBuffer
0.5μlT4ligase(NEB，M0202S)
4.5 μ l aqua sterilisas
Hatch 1 hour for 16 DEG C.
4, connection product conversion DH5 α competent cell (TransGen, CD201) above-mentioned steps obtained also is coated with Amp+ flat board (50 μ g/ml), and picked clones.
5, with the universal primer U6 such as shown in sequence table SEQ IDNO.9, positive colony is obtained with the method qualification of routine order-checking.
6,37 DEG C of shaking tables shake bacterium incubated overnight positive colony, and with AxyPrepPlasmidMiniprepKit(AP-MN-P-250) extracting plasmid, obtain pGL3-U6-hPD1sg1 plasmid (as shown in sequence table SEQ IDNO.14).
7, cell cultures and transfection
(1) HEK293T cell inoculation culture is in the high sugared nutrient solution of DMEM (HyClone, SH30022.01B), wherein containing 10%FBS, penicillin(100U/ml) and streptomycin(100 μ g/ml).
(2) divide in 12 orifice plates before transfection, when 70% ~ 80% density, carry out transfection.
(3) according to Lipofectamine tM2000TransfectionReagent(Invitrogen, operational manual 11668-019), by 0.5 μ gpGL3-U6-hPD1sg1(as shown in sequence table SEQ IDNO.14) mix with the pST1374-NLS-flag-Cas9-ZF plasmid (as shown in sequence table SEQ IDNO.11) of 1.5 μ g, in cotransfection to every porocyte, change liquid after 6 ~ 8 hours, and add Blasticidin(Sigma, 15205) and Puromycin(Merck, 540411) medicine sieve, collected cell after 48 hours.
The preparation method of plasmid pST1374-NLS-flag-Cas9-ZF is see document: Shenetal.2013, Generationofgene-modifiedmiceviaCas9/RNA-mediatedgenetar geting.CellResearch23,720-723.(doi:10.1038/cr.2013.46)
8, T7EN1 enzyme cuts detection
(1), after the cell collected being digested with 100 μ g/ml Proteinase K cracking in lysate (10 μMs of Tris-HCl, 0.4MNaCl, 2 μMs of EDTA, 1%SDS), be dissolved into after phenol-chloroform extracting in 50 μ l deionized waters.
(2) primer hPD1testFor and hPD1testRev of sequence as SEQIDNO.12 and SEQIDNO.13 is used to carry out pcr amplification, purify with AxyPrepPCRcleanup and obtain PCR recovery product, get that 200ng is unified to be diluted to 20 μ l and to carry out sex change, annealing, program as: 95 DEG C, 5min; 95 – 85 DEG C at-2 DEG C/s; 85 – 25 DEG C at-0.1 DEG C/s; Holdat4 DEG C.
(3) in 20 μ l systems, add T7EN10.3 μ l, after 37 DEG C of enzymes cut 30 minutes, add 2 μ l10XLoadingBuffer, the agarose gel electrophoresis with 2.5% detects.
9, TA cloning and sequencing
(1) T7EN1 enzyme is cut PCR that detecting step (2) obtains to reclaim product rTaq and carry out adding A reaction.Adding A reaction system is:
700 ~ 800ngPCR reclaims product
5μl10XBuffer(Mg 2+free)
3μlMg 2+
4μldNTP
0.5μlrTaq(TAKARA,R001AM)
Moisturizing to 50 μ l system.
37 DEG C of incubations are after 30 minutes, get 1 μ l product and pMD19-Tvector (TAKARA, 3271) connects and transforms DH5 α competent cell (TransGen, CD201).
(2) picking mono-clonal is with sequence as the universal primer U6 of sequence table SEQ IDNO.9 checks order, and finds according to sequencing result (as shown in sequence table SEQ IDNO.18): target gene PD1 has lacked one section of sequence of sgRNA target, gene knockout success.
Embodiment 3 utilizes CRISPR-Cas9 specific knockdown people PD1 gene (for the sgRNA of target PD1 gene as shown in sequence table SEQ IDNO.82)
1, the pGL3-U6-sgRNA plasmid of linearizing sequence as shown in sequence table SEQ IDNO.10.
Enzyme cuts system and condition is as follows:
2μgpGL3-U6-sgRNA（400ng/μl）；
1μlCutSmartBuffer；
1μlBsaI(NEB，R0535L)；
Moisturizing to 50 μ l, hatches 3 ~ 4 hours for 37 DEG C, vibrates at set intervals and centrifugal in case droplet evaporation covers to pipe.
Enzyme cuts into rear AxyPrepPCRCleanupKit(AP-PCR-250) purifying is recycled in 20 ~ 40 μ l aqua sterilisas.
2, by the double-strand sgRNA oligonucleotide (itself Forwardoligo with Reverseoligo sequence is respectively if sequence table SEQ IDNO.3 is with 4 Suo Shi) that can be connected into U6 carrier for expression of eukaryon of acquisition after sex change, annealing, being connected with linearizing pGL3-U6-sgRNA plasmid obtains pGL3-U6-hPD1sg1 plasmid.
Linked system is as follows:
3 μ l50 μMs of annealed product double-strand sgRNA oligonucleotides, its forwardoligo is as shown in sequence table SEQ IDNO.3, and its reverseoligo is as shown in sequence table SEQ IDNO.4)
The linearizing pGL3-U6-sgRNA plasmid of 1 μ l (25ng/ μ l)
1μlT4ligationBuffer
0.5μlT4ligase(NEB，M0202S)
4.5 μ l aqua sterilisas
Hatch 1 hour for 16 DEG C.
4, connection product conversion DH5 α competent cell (TransGen, CD201) above-mentioned steps obtained also is coated with Amp+ flat board (50 μ g/ml), and picked clones.
5, with the universal primer U6 such as shown in sequence table SEQ IDNO.9, positive colony is obtained with the method qualification of routine order-checking.
6,37 DEG C of shaking tables shake bacterium incubated overnight positive colony, and with AxyPrepPlasmidMiniprepKit(AP-MN-P-250) extracting plasmid, obtain pGL3-U6-hPD1sg2 plasmid (as shown in sequence table SEQ IDNO.15).
7, cell cultures and transfection
(1) HEK293T cell inoculation culture is in the high sugared nutrient solution of DMEM (HyClone, SH30022.01B), wherein containing 10%FBS, penicillin(100U/ml) and streptomycin(100 μ g/ml).
(2) divide in 12 orifice plates before transfection, when 70% ~ 80% density, carry out transfection.
(3) according to Lipofectamine tM2000TransfectionReagent(Invitrogen, operational manual 11668-019), the pST1374-NLS-flag-Cas9-ZF plasmid (as shown in sequence table SEQ IDNO.11) of 0.5 μ gpGL3-U6-hPD1sg2 plasmid (as shown in sequence table SEQ IDNO.15) with 1.5 μ g is mixed, in cotransfection to every porocyte, change liquid after 6 ~ 8 hours, and add Blasticidin(Sigma, 15205) and Puromycin(Merck, 540411) medicine sieve, collected cell after 48 hours.
The preparation method of plasmid pST1374-NLS-flag-Cas9-ZF is see document: Shenetal.2013, Generationofgene-modifiedmiceviaCas9/RNA-mediatedgenetar geting.CellResearch23,720-723.(doi:10.1038/cr.2013.46)
8, T7EN1 enzyme cuts detection
(1), after the cell collected being digested with 100 μ g/ml Proteinase K cracking in lysate (10 μMs of Tris-HCl, 0.4MNaCl, 2 μMs of EDTA, 1%SDS), be dissolved into after phenol-chloroform extracting in 50 μ l deionized waters.
(2) primer hPD1testFor and hPD1testRev of sequence as SEQIDNO.12 and SEQIDNO.13 is used to carry out pcr amplification, purify with AxyPrepPCRcleanup and obtain PCR recovery product, get that 200ng is unified to be diluted to 20 μ l and to carry out sex change, annealing, program as: 95 DEG C, 5min; 95 – 85 DEG C at-2 DEG C/s; 85 – 25 DEG C at-0.1 DEG C/s; Holdat4 DEG C.
(3) in 20 μ l systems, add T7EN10.3 μ l, after 37 DEG C of enzymes cut 30 minutes, add 2 μ l10XLoadingBuffer, the agarose gel electrophoresis with 2.5% detects.
9, TA cloning and sequencing
(1) T7EN1 enzyme is cut PCR that detecting step (2) obtains to reclaim product rTaq and carry out adding A reaction.Adding A reaction system is:
700 ~ 800ngPCR reclaims product
5μl10XBuffer(Mg 2+free)
3μlMg 2+
4μldNTP
0.5μlrTaq(TAKARA,R001AM)
Moisturizing to 50 μ l system.
37 DEG C of incubations are after 30 minutes, get 1 μ l product and pMD19-Tvector (TAKARA, 3271) connects and transforms DH5 α competent cell (TransGen, CD201).
(2) picking mono-clonal is with sequence as the universal primer U6 of sequence table SEQ IDNO.9 checks order, and finds according to sequencing result (as shown in sequence table SEQ IDNO.19): target gene PD1 has lacked one section of sequence of sgRNA target, gene knockout success.
Embodiment 4 utilizes CRISPR-Cas9 specific knockdown people PD1 gene (for the sgRNA of target PD1 gene as shown in sequence table SEQ IDNO.57)
1, the pGL3-U6-sgRNA plasmid of linearizing sequence as shown in sequence table SEQ IDNO.10.
Enzyme cuts system and condition is as follows:
2μgpGL3-U6-sgRNA（400ng/μl）；
1μlCutSmartBuffer；
1μlBsaI(NEB，R0535L)；
Moisturizing to 50 μ l, hatches 3 ~ 4 hours for 37 DEG C, vibrates at set intervals and centrifugal in case droplet evaporation covers to pipe.
Enzyme cuts into rear AxyPrepPCRCleanupKit(AP-PCR-250) purifying is recycled in 20 ~ 40 μ l aqua sterilisas.
2, by the double-strand sgRNA oligonucleotide (itself Forwardoligo with Reverseoligo sequence is respectively if sequence table SEQ IDNO.5 is with 6 Suo Shi) that can be connected into U6 carrier for expression of eukaryon of acquisition after sex change, annealing, being connected with linearizing pGL3-U6-sgRNA plasmid obtains pGL3-U6-hPD1sg1 plasmid.
Linked system is as follows:
3 μ l50 μM annealed product (double-strand sgRNA oligonucleotide, its forwardoligo is as shown in sequence table SEQ IDNO.5, and its reverseoligo is as shown in sequence table SEQ IDNO.6)
The linearizing pGL3-U6-sgRNA plasmid of 1 μ l (25ng/ μ l)
1μlT4ligationBuffer
0.5μlT4ligase(NEB，M0202S)
4.5 μ l aqua sterilisas
Hatch 1 hour for 16 DEG C.
4, connection product conversion DH5 α competent cell (TransGen, CD201) above-mentioned steps obtained also is coated with Amp+ flat board (50 μ g/ml), and picked clones.
5, with the universal primer U6 such as shown in sequence table SEQ IDNO.9, positive colony is obtained with the method qualification of routine order-checking.
6,37 DEG C of shaking tables shake bacterium incubated overnight positive colony, and with AxyPrepPlasmidMiniprepKit(AP-MN-P-250) extracting plasmid, obtain pGL3-U6-hPD1sg3(as shown in sequence table SEQ IDNO.16).
7, cell cultures and transfection
(1) HEK293T cell inoculation culture is in the high sugared nutrient solution of DMEM (HyClone, SH30022.01B), wherein containing 10%FBS, penicillin(100U/ml) and streptomycin(100 μ g/ml).
(2) divide in 12 orifice plates before transfection, when 70% ~ 80% density, carry out transfection.
(3) according to Lipofectamine tM2000TransfectionReagent(Invitrogen, operational manual 11668-019), the pST1374-NLS-flag-Cas9-ZF plasmid (as shown in sequence table SEQ IDNO.10) of 0.5 μ gpGL3-U6-hPD1sg3 plasmid (as shown in sequence table SEQ IDNO.16) with 1.5 μ g is mixed, in cotransfection to every porocyte, change liquid after 6 ~ 8 hours, and add Blasticidin(Sigma, 15205) and Puromycin(Merck, 540411) medicine sieve, collected cell after 48 hours.
The preparation method of plasmid pST1374-NLS-flag-Cas9-ZF is see document: Shenetal.2013, Generationofgene-modifiedmiceviaCas9/RNA-mediatedgenetar geting.CellResearch23,720-723.(doi:10.1038/cr.2013.46)
8, T7EN1 enzyme cuts detection
(1), after the cell collected being digested with 100 μ g/ml Proteinase K cracking in lysate (10 μMs of Tris-HCl, 0.4MNaCl, 2 μMs of EDTA, 1%SDS), be dissolved into after phenol-chloroform extracting in 50 μ l deionized waters.
(2) primer hPD1testFor and hPD1testRev of sequence as SEQIDNO.12 and SEQIDNO.13 is used to carry out pcr amplification, purify with AxyPrepPCRcleanup and obtain PCR recovery product, get that 200ng is unified to be diluted to 20 μ l and to carry out sex change, annealing, program as: 95 DEG C, 5min; 95 – 85 DEG C at-2 DEG C/s; 85 – 25 DEG C at-0.1 DEG C/s; Holdat4 DEG C.
(3) in 20 μ l systems, add T7EN10.3 μ l, after 37 DEG C of enzymes cut 30 minutes, add 2 μ l10XLoadingBuffer, the agarose gel electrophoresis with 2.5% detects.
9, TA cloning and sequencing
(1) T7EN1 enzyme is cut PCR that detecting step (2) obtains to reclaim product rTaq and carry out adding A reaction.Adding A reaction system is:
700 ~ 800ngPCR reclaims product
5μl10XBuffer(Mg 2+free)
3μlMg 2+
4μldNTP
0.5μlrTaq(TAKARA,R001AM)
Moisturizing to 50 μ l system.
37 DEG C of incubations are after 30 minutes, get 1 μ l product and pMD19-Tvector (TAKARA, 3271) connects and transforms DH5 α competent cell (TransGen, CD201).
(2) picking mono-clonal is with sequence as the universal primer U6 of sequence table SEQ IDNO.9 checks order, and finds according to sequencing result (as shown in sequence table SEQ IDNO.20): target gene PD1 lacks the sequence of sgRNA target, gene knockout success.
Embodiment 5 utilizes CRISPR-Cas9 specific knockdown people PD1 gene (for the sgRNA of target PD1 gene as shown in sequence table SEQ IDNO.78)
1, the pGL3-U6-sgRNA plasmid of linearizing sequence as shown in sequence table SEQ IDNO.10.
Enzyme cuts system and condition is as follows:
2μgpGL3-U6-sgRNA（400ng/μl）；
1μlCutSmartBuffer；
1μlBsaI(NEB，R0535L)；
Moisturizing to 50 μ l, hatches 3 ~ 4 hours for 37 DEG C, vibrates at set intervals and centrifugal in case droplet evaporation covers to pipe.
Enzyme cuts into rear AxyPrepPCRCleanupKit(AP-PCR-250) purifying is recycled in 20 ~ 40 μ l aqua sterilisas.
2, by the double-strand sgRNA oligonucleotide (itself Forwardoligo with Reverseoligo sequence is respectively if sequence table SEQ IDNO.7 is with 8 Suo Shi) that can be connected into U6 carrier for expression of eukaryon of acquisition after sex change, annealing, being connected with linearizing pGL3-U6-sgRNA plasmid obtains pGL3-U6-hPD1sg4 plasmid.
Linked system is as follows:
3 μ l50 μM annealed product (double-strand sgRNA oligonucleotide, its forwardoligo is as shown in sequence table SEQ IDNO.7, and its reverseoligo is as shown in sequence table SEQ IDNO.8)
The linearizing pGL3-U6-sgRNA plasmid of 1 μ l (25ng/ μ l)
1μlT4ligationBuffer
0.5μlT4ligase(NEB，M0202S)
4.5 μ l aqua sterilisas
Hatch 1 hour for 16 DEG C.
3, connection product conversion DH5 α competent cell (TransGen, CD201) above-mentioned steps obtained also is coated with Amp+ flat board (50 μ g/ml), and picked clones.
4, with the universal primer U6 such as shown in sequence table SEQ IDNO.9, positive colony is obtained with the method qualification of routine order-checking.
5,37 DEG C of shaking tables shake bacterium incubated overnight positive colony, and with AxyPrepPlasmidMiniprepKit(AP-MN-P-250) extracting plasmid, obtain pGL3-U6-hPD1sg4(as shown in sequence table SEQ IDNO.17).
6, cell cultures and transfection
(1) HEK293T cell inoculation culture is in the high sugared nutrient solution of DMEM (HyClone, SH30022.01B), wherein containing 10%FBS, penicillin(100U/ml) and streptomycin(100 μ g/ml).
(2) divide in 12 orifice plates before transfection, when 70% ~ 80% density, carry out transfection.
(3) according to Lipofectamine tM2000TransfectionReagent(Invitrogen, operational manual 11668-019), by 0.5 μ gpGL3-U6-hPD1sg4(as shown in sequence table SEQ IDNO.17) mix with the pST1374-NLS-flag-Cas9-ZF plasmid (as shown in sequence table SEQ IDNO.11) of 1.5 μ g, in cotransfection to every porocyte, change liquid after 6 ~ 8 hours, and add Blasticidin(Sigma, 15205) and Puromycin(Merck, 540411) medicine sieve, collected cell after 48 hours.
The preparation method of plasmid pST1374-NLS-flag-Cas9-ZF is see document: Shenetal.2013, Generationofgene-modifiedmiceviaCas9/RNA-mediatedgenetar geting.CellResearch23,720-723.(doi:10.1038/cr.2013.46)
8, T7EN1 enzyme cuts detection
(1), after the cell collected being digested with 100 μ g/ml Proteinase K cracking in lysate (10 μMs of Tris-HCl, 0.4MNaCl, 2 μMs of EDTA, 1%SDS), be dissolved into after phenol-chloroform extracting in 50 μ l deionized waters.
(2) primer hPD1testFor and hPD1testRev of sequence as SEQIDNO.12 and SEQIDNO.13 is used to carry out pcr amplification, purify with AxyPrepPCRcleanup and obtain PCR recovery product, get that 200ng is unified to be diluted to 20 μ l and to carry out sex change, annealing, program as: 95 DEG C, 5min; 95 – 85 DEG C at-2 DEG C/s; 85 – 25 DEG C at-0.1 DEG C/s; Holdat4 DEG C.
(3) in 20 μ l systems, add T7EN10.3 μ l, after 37 DEG C of enzymes cut 30 minutes, add 2 μ l10XLoadingBuffer, the agarose gel electrophoresis with 2.5% detects.
9, TA cloning and sequencing
(1) T7EN1 enzyme is cut PCR that detecting step (2) obtains to reclaim product rTaq and carry out adding A reaction.Adding A reaction system is:
700 ~ 800ngPCR reclaims product
5μl10XBuffer(Mg 2+free)
3μlMg 2+
4μldNTP
0.5μlrTaq(TAKARA,R001AM)
Moisturizing to 50 μ l system.
37 DEG C of incubations are after 30 minutes, get 1 μ l product and pMD19-Tvector (TAKARA, 3271) connects and transforms DH5 α competent cell (TransGen, CD201).
(2) picking mono-clonal is with sequence as the universal primer U6 of sequence table SEQ IDNO.9 checks order, and finds according to sequencing result (as shown in sequence table SEQ IDNO.21): target gene PD1 lacks the sequence of sgRNA target, gene knockout success.
Embodiment 6 utilizes CRISPR-Cas9 specific knockdown people PD1 gene
SgRNA for target PD1 gene is two sgRNA targets altogether, and its sequence is as shown in sequence table SEQ IDNO.52 and 82, and the target initiation site of these two sgRNA on PD1 gene, at a distance of 5bp, can significantly improve and knock out efficiency.
1, the pGL3-U6-sgRNA plasmid of linearizing sequence as shown in sequence table SEQ IDNO.10.
Enzyme cuts system and condition is as follows:
2μgpGL3-U6-sgRNA（400ng/μl）；
1μlCutSmartBuffer；
1μlBsaI(NEB，R0535L)；
Moisturizing to 50 μ l, hatches 3 ~ 4 hours for 37 DEG C, vibrates at set intervals and centrifugal in case droplet evaporation covers to pipe.
Enzyme cuts into rear AxyPrepPCRCleanupKit(AP-PCR-250) purifying is recycled in 20 ~ 40 μ l aqua sterilisas.
2, by the double-strand sgRNA oligonucleotide (itself Forwardoligo with Reverseoligo sequence is respectively if sequence table SEQ IDNO.1 is with 2 Suo Shi) that can be connected into U6 carrier for expression of eukaryon of acquisition after sex change, annealing, being connected with linearizing pGL3-U6-sgRNA plasmid obtains pGL3-U6-hPD1sg1 plasmid.
By the double-strand sgRNA oligonucleotide (itself Forwardoligo with Reverseoligo sequence is respectively if sequence table SEQ IDNO.3 is with 4 Suo Shi) that can be connected into U6 carrier for expression of eukaryon of acquisition after sex change, annealing, being connected with linearizing pGL3-U6-sgRNA plasmid obtains pGL3-U6-hPD1sg2 plasmid.
Linked system is as follows:
3 μ l50 μM annealed product (double-strand sgRNA oligonucleotides, its forwardoligo is as shown in sequence table SEQ IDNO.1, its reverseoligo is as shown in sequence table SEQ IDNO.2) or (double-strand sgRNA oligonucleotide, its forwardoligo is as shown in sequence table SEQ IDNO.3, and its reverseoligo is as shown in sequence table SEQ IDNO.4)
The linearizing pGL3-U6-sgRNA plasmid of 1 μ l (25ng/ μ l)
1μlT4ligationBuffer
0.5μlT4ligase(NEB，M0202S)
4.5 μ l aqua sterilisas
Hatch 1 hour for 16 DEG C.
4, the connection product that above-mentioned steps obtains is transformed DH5 α competent cell (TransGen, CD201) respectively and is coated with Amp+ flat board (50 μ g/ml), and picked clones.
5, with the universal primer U6 such as shown in sequence table SEQ IDNO.9, positive colony is obtained with the method qualification of routine order-checking.
6,37 DEG C of shaking tables shake bacterium incubated overnight positive colony, and with AxyPrepPlasmidMiniprepKit(AP-MN-P-250) extracting plasmid, obtain pGL3-U6-hPD1sg1(as shown in sequence table SEQ IDNO.14) and pGL3-U6-hPD1sg2(as shown in sequence table SEQ IDNO.15).
7, cell cultures and transfection
(1) HEK293T cell inoculation culture is in the high sugared nutrient solution of DMEM (HyClone, SH30022.01B), wherein containing 10%FBS, penicillin(100U/ml) and streptomycin(100 μ g/ml).
(2) divide in 12 orifice plates before transfection, when 70% ~ 80% density, carry out transfection.
(3) according to Lipofectamine tM2000TransfectionReagent(Invitrogen, operational manual 11668-019), by 0.5 μ gpGL3-U6-hPD1sg1(as shown in sequence table SEQ IDNO.14) and 0.5 μ gpGL3-U6-hPD1sg2(as shown in sequence table SEQ IDNO.15) mix with the pST1374-NLS-flag-Cas9-ZF plasmid (as shown in sequence table SEQ IDNO.11) of 1.5 μ g, in cotransfection to every porocyte, liquid is changed after 6 ~ 8 hours, and add Blasticidin(Sigma, 15205) and Puromycin(Merck, 540411) medicine sieve, collected cell after 48 hours.
The preparation method of plasmid pST1374-NLS-flag-Cas9-ZF is see document: Shenetal.2013, Generationofgene-modifiedmiceviaCas9/RNA-mediatedgenetar geting.CellResearch23,720-723.(doi:10.1038/cr.2013.46)
8, T7EN1 enzyme cuts detection
(1), after the cell collected being digested with 100 μ g/ml Proteinase K cracking in lysate (10 μMs of Tris-HCl, 0.4MNaCl, 2 μMs of EDTA, 1%SDS), be dissolved into after phenol-chloroform extracting in 50 μ l deionized waters.
(2) primer hPD1testFor and hPD1testRev of sequence as SEQIDNO.12 and SEQIDNO.13 is used to carry out pcr amplification, purify with AxyPrepPCRcleanup and obtain PCR recovery product, get that 200ng is unified to be diluted to 20 μ l and to carry out sex change, annealing, program as: 95 DEG C, 5min; 95 – 85 DEG C at-2 DEG C/s; 85 – 25 DEG C at-0.1 DEG C/s; Holdat4 DEG C.
(3) in 20 μ l systems, add T7EN10.3 μ l, after 37 DEG C of enzymes cut 30 minutes, add 2 μ l10XLoadingBuffer, the agarose gel electrophoresis with 2.5% detects.
As shown in Figure 2, can be found by agarose gel electrophoresis: occur broken ends connect the genome repaired can because with protogene group Incomplete matching, and to be cut by T7EN1.Demonstrate less band.
9, TA cloning and sequencing
(1) T7EN1 enzyme is cut PCR that detecting step (2) obtains to reclaim product rTaq and carry out adding A reaction.Adding A reaction system is:
700 ~ 800ngPCR reclaims product
5μl10XBuffer(Mg 2+free)
3μlMg 2+
4μldNTP
0.5μlrTaq(TAKARA,R001AM)
Moisturizing to 50 μ l system.
37 DEG C of incubations are after 30 minutes, get 1 μ l product and pMD19-Tvector (TAKARA, 3271) connects and transforms DH5 α competent cell (TransGen, CD201).
(2) picking mono-clonal with sequence as the universal primer U6 of sequence table SEQ IDNO.9 checks order, find according to sequencing result (as shown in sequence table SEQ IDNO.22): target gene PD1 has lacked one section, the centre of two sgRNA target sequences, gene knockout success (as shown in Figure 3).
Embodiment 7 utilizes CRISPR-Cas9 specific knockdown people PD1 gene
SgRNA for target PD1 gene is two sgRNA targets altogether, and its sequence is as shown in sequence table SEQ IDNO.57 and 78, and the target initiation site of these two sgRNA on PD1 gene, at a distance of 8bp, can significantly improve and knock out efficiency.
1, the pGL3-U6-sgRNA plasmid of linearizing sequence as shown in sequence table SEQ IDNO.10.
Enzyme cuts system and condition is as follows:
2μgpGL3-U6-sgRNA（400ng/μl）；
1μlCutSmartBuffer；
1μlBsaI(NEB，R0535L)；
Moisturizing to 50 μ l, hatches 3 ~ 4 hours for 37 DEG C, vibrates at set intervals and centrifugal in case droplet evaporation covers to pipe.
Enzyme cuts into rear AxyPrepPCRCleanupKit(AP-PCR-250) purifying is recycled in 20 ~ 40 μ l aqua sterilisas.
2, by the double-strand sgRNA oligonucleotide (itself Forwardoligo with Reverseoligo sequence is respectively if sequence table SEQ IDNO.5 is with 6 Suo Shi) that can be connected into U6 carrier for expression of eukaryon of acquisition after sex change, annealing, being connected with linearizing pGL3-U6-sgRNA plasmid obtains pGL3-U6-hPD1sg3 plasmid.
By the double-strand sgRNA oligonucleotide (itself Forwardoligo with Reverseoligo sequence is respectively if sequence table SEQ IDNO.7 is with 8 Suo Shi) that can be connected into U6 carrier for expression of eukaryon of acquisition after sex change, annealing, being connected with linearizing pGL3-U6-sgRNA plasmid obtains pGL3-U6-hPD1sg4 plasmid.
Linked system is as follows:
3 μ l50 μM annealed product (sgRNA oligonucleotides, its forwardoligo is as shown in sequence table SEQ IDNO.5, its reverseoligo is as shown in sequence table SEQ IDNO.6) or (sgRNA oligonucleotide, its forwardoligo is as shown in sequence table SEQ IDNO.7, and its reverseoligo is as shown in sequence table SEQ IDNO.8)
The linearizing pGL3-U6-sgRNA plasmid of 1 μ l (25ng/ μ l)
1μlT4ligationBuffer
0.5μlT4ligase(NEB，M0202S)
4.5 μ l aqua sterilisas
Hatch 1 hour for 16 DEG C.
4, connection product difference transformed competence colibacillus cell DH5 α (TransGen, CD201) above-mentioned steps obtained also is coated with Amp+ flat board (50 μ g/ml), and picked clones.
5, with the universal primer U6 such as shown in sequence table SEQ IDNO.9, positive colony is obtained with the method qualification of routine order-checking.
6,37 DEG C of shaking tables shake bacterium incubated overnight positive colony, and with AxyPrepPlasmidMiniprepKit(AP-MN-P-250) extracting plasmid, obtain pGL3-U6-hPD1sg3 plasmid (as shown in sequence table SEQ IDNO.16) and pGL3-U6-hPD1sg4 plasmid (as shown in sequence table SEQ IDNO.17).
7, cell cultures and transfection
(1) HEK293T cell inoculation culture is in the high sugared nutrient solution of DMEM (HyClone, SH30022.01B), wherein containing 10%FBS, penicillin(100U/ml) and streptomycin(100 μ g/ml).
(2) divide in 12 orifice plates before transfection, when 70% ~ 80% density, carry out transfection.
(3) according to Lipofectamine tM2000TransfectionReagent(Invitrogen, operational manual 11668-019), 0.5 μ gpGL3-U6-hPD1sg3 plasmid (as shown in sequence table SEQ IDNO.16) and 0.5 μ gpGL3-U6-hPD1sg4 plasmid (as shown in sequence table SEQ IDNO.17) are mixed with the pST1374-NLS-flag-Cas9-ZF plasmid (as shown in sequence table SEQ IDNO.11) of 1.5 μ g, in cotransfection to every porocyte, liquid is changed after 6 ~ 8 hours, and add Blasticidin(Sigma, 15205) and Puromycin(Merck, 540411) medicine sieve, cell is collected after 48 hours.
The preparation method of plasmid pST1374-NLS-flag-Cas9-ZF is see document: Shenetal.2013, Generationofgene-modifiedmiceviaCas9/RNA-mediatedgenetar geting.CellResearch23,720-723.(doi:10.1038/cr.2013.46)
8, T7EN1 enzyme cuts detection
(1), after the cell collected being digested with 100 μ g/ml Proteinase K cracking in lysate (10 μMs of Tris-HCl, 0.4MNaCl, 2 μMs of EDTA, 1%SDS), be dissolved into after phenol-chloroform extracting in 50 μ l deionized waters.
(2) primer hPD1testFor and hPD1testRev of sequence as SEQIDNO.12 and SEQIDNO.13 is used to carry out pcr amplification, purify with AxyPrepPCRcleanup and obtain PCR recovery product, get that 200ng is unified to be diluted to 20 μ l and to carry out sex change, annealing, program as: 95 DEG C, 5min; 95 – 85 DEG C at-2 DEG C/s; 85 – 25 DEG C at-0.1 DEG C/s; Holdat4 DEG C.
(3) in 20 μ l systems, add T7EN10.3 μ l, after 37 DEG C of enzymes cut 30 minutes, add 2 μ l10XLoadingBuffer, the agarose gel electrophoresis with 2.5% detects.
As shown in Figure 2, can be found by agarose gel electrophoresis: occur broken ends connect the genome repaired can because with protogene group Incomplete matching, and to be cut by T7EN1.Demonstrate less band.
9, TA cloning and sequencing
(1) T7EN1 enzyme is cut PCR that detecting step (2) obtains to reclaim product rTaq and carry out adding A reaction.Adding A reaction system is:
700 ~ 800ngPCR reclaims product
5μl10XBuffer(Mg 2+free)
3μlMg 2+
4μldNTP
0.5μlrTaq(TAKARA,R001AM)
Moisturizing to 50 μ l system.
37 DEG C of incubations are after 30 minutes, get 1 μ l product and pMD19-Tvector (TAKARA, 3271) connects and transformed competence colibacillus cell DH5 α (TransGen, CD201).
(2) picking mono-clonal with sequence as the universal primer U6 of sequence table SEQ IDNO.9 checks order, find according to sequencing result (as shown in sequence table SEQ IDNO.23): target gene PD1 has lacked one section, the centre of two sgRNA target sequences, gene knockout success (as shown in Figure 3).

Claims (1)

  1. The method of 1.CRISPR-Cas9 specific knockdown people PD1 gene, the method is used for non-diagnostic or therapeutic purpose, it is characterized by and comprises the steps:
    (1) sgRNA is provided, the target sequence of described sgRNA on PD1 gene meets the series arrangement rule of 5 '-N (21) GG, the target sequence of described sgRNA on PD1 gene is positioned at the exon of gene, the target sequence of described sgRNA on PD1 gene is positioned on the common exon of different various shear-forms, the target sequence of described sgRNA on PD1 gene is unique, and the target site sequence of described sgRNA on PD1 is as sequence table SEQ IDNO.52, 57, shown in 78 or 82 any sequences, add that CCGG synthesis obtains forward oligonucleotide and Forwardoligo at 5 ' of the target site sequence of described sgRNA on PD1, obtain the complementary strand of the target site sequence of sgRNA on PD1, and add that AAAC synthesis obtains reverse oligonucleotide and Reverseoligo at 5 ' of complementary strand, by 1 of synthesis to the paired sex change of forwardoligo and reverseoligo of the sgRNA oligonucleotide of complementation, annealing, after annealing, form the double-strand sgRNA oligonucleotide that can be connected into U6 carrier for expression of eukaryon,
    (2) the pGL3-U6-sgRNA plasmid of linearizing sequence as shown in sequence table SEQ IDNO.10; The double-strand sgRNA oligonucleotide of annealing is connected with linearizing pGL3-U6-sgRNA plasmid and obtains pGL3-U6-hPD1sg plasmid; PGL3-U6-hPD1sg Plastid transformation competence bacterium is also coated with Amp+ flat board, and picking mono-clonal also identifies positive colony with the universal primer U6 of sequence as shown in sequence table SEQ IDNO.9 by order-checking; 37 DEG C of shaking tables shake positive colony bacterium and spend the night and use article No. to be the AxyPrepPlasmidMiniprepKit extracting pGL3-U6-hPD1sg plasmid of AP-MN-P-250;
    (3) Lipofectamine is used tM2000TransfectionReagent loading pGL3-U6-hPD1sg plasmid and sequence are the pST1374-NLS-flag-Cas9-ZF plasmid of SEQIDNO.11, cotransfection cell;
    (4) cut with T7EN1 enzyme and to detect and TA cloning and sequencing confirmation PD1 gene has been knocked and has obtained the cell of gene knockout.
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