CN106801056A - The slow virus carrier and application of a kind of sgRNA and its structure - Google Patents
The slow virus carrier and application of a kind of sgRNA and its structure Download PDFInfo
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Abstract
The present invention relates to field of gene, more particularly to a kind of sgRNA and its structure slow virus carrier and application, specially with SIVmac1A11Slow virus is skeleton, the sgRNA of expression SpCas9 albumen and gene specific, for treating people and simian acquired immunodeficiency syndrome.The nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1 2.Present invention employs most efficient CRISPR/Cas9 gene editings instrument at present, designed CXCR4/CCR5 gene sgRNA sites have the gene knockout activity better than other sites of conventional research institute report, the gene therapy of to be also first Application infect in SIV rhesus macaque, has the advantage such as simple operation, with low cost compared to ZFN and TALEN.
Description
Technical field
The present invention relates to field of gene, more particularly to a kind of sgRNA and its structure slow virus carrier and application, tool
Body is with SIVmac1A11Slow virus is skeleton, the sgRNA of expression SpCas9 albumen and gene specific, for treating people and monkey
AIDS.
Background technology
The short palindrome repetitive sequence of regular intervals of cluster and its Cas9 protein systems (CRISPR/Cas9) of correlation are bacteriums
With the natural immunology defense that archeobacteria is used to resist exogenous virus infection.After the DNA invasion bacterium/archeobacterias of external source, can be thin
The RNA homing sequence (guideRNA) complementary with exogenous DNA specific region identification in born of the same parents, and guide Cas9 nucleases to reach identification
Position, digestion is carried out to target sequence, so that exogenous DNA of degrading.This system concrete operating principle is as follows：crRNA(CRISPR-
Derived RNA) tracrRNA/ is combined to form with tracrRNA (trans-activating RNA) by base pairing
CrRNA compounds, the compound can guide nuclease Cas9 albumen double in the target sequence target site shearing matched with crRNA
Chain DNA.By both RNA of engineer, the single-stranded sgRNA (singleguide to be formed with guiding function can be transformed
RNA), guiding Cas9 carries out fixed point cutting to DNA.
As the double-stranded DNA associated proteins that a kind of RNA is oriented to, Cas9 effector nucleases be known first unification because
Sub (unifying factor), can common location RNA, DNA and albumen, so as to possess huge transformation potentiality.By albumen and nothing
Cas9 (Cas9nuclease-null) fusions of nuclease, and appropriate sgRNA is expressed, any dsDNA sequences can be targeted, and
The end of sgRNA may be connected to target dna, and the combination of Cas9 is not influenceed.Therefore, Cas9 can bring at any dsDNA sequences
Any fusion protein and RNA, this brings great potential for the research and transformation of organism.Therefore CRISPR/Cas9 conducts quickly
New gene editing instrument is widely used in the fields such as genetic engineering, crop breeding and gene therapy.
AIDS is a kind of chronic infectious disease for being difficult to and effecting a radical cure.Although joint ART can control HIV-
The duplication of 1 virus, maintains the existence of patient, but the problems such as side effects of pharmaceutical drugs and the resistance to the action of a drug of long-term prescription generation still
In the presence of searching is more effective, and can improve emphasis and difficult point that the therapeutic strategy of life in patients is AIDS research field.Chinese mugwort
The gene therapy for growing disease has been directed to the whole life cycle of viral infection host, wherein targeting poisoning intrusion door --- auxiliary
The therapeutic strategy of receptor CXCR 4 and CCR5 clinically presents dawn.Infected by HIV -1 in 2007 and suffer from acute
The man of myelocytic leukemia, by the confession of 32 bases of heteroplastic transplantation CCR5 genes natural deletions (Δ 32 of CCR5 Δs 32/)
After person's marrow, the bounce-back of internal virus is still not detected by so far, and health is good, it is that global first case is cured to be recognized
AIDS patient.Follow-up research shows that the donor bone marrow of phenotype is the key for realizing radical cure.But naturally carry CCR5 Δs
The number of the genotype of 32/ Δ 32 is rare, and there are problems that HLA, limits the popularization of this therapy.In addition, there will be a large amount of
Research confirms that HIV-1 different strains has different co-receptor preferendum (CCR5 preferendums, CXCR4 preferendums, the double preferendums of X4-R5
Deng), and the HIV-1 variants of different preferendums are there is also in same patient's body.Therefore patient is modified using genetic tool
The genes such as itself CCR5, CXCR4, and autologous feedback, for controlling and treating HIV-1 infection, it is possible to finally realize feature
Cure.
The A of CN 104480144 disclose a kind of CRISPR/Cas9 recombinant slow virus that can be used for AIDS gene therapy and carry
Body and its slow virus, the recombined lentivirus vector by slow virus carrier lentiCRISPR with BsmBI digestions after, be connected into band BsmBI
The CXCR4 specific target sequences of cohesive end are recombinated and obtained.Its CRISPR/Cas9 recombined lentivirus vector for using people, its
Security is not high.
Rhesus macaque as AIDS research model, with the advantage that mouse does not have：Monkey carries on the back with the heredity of people first
Scape closer to；Next, research has proven to HIV-1 and originates from SIV, and the course of disease and pathological characters of HIV-1 infection people infect with SIV
Rhesus macaque is quite similar.Therefore, felt for clinical treatment HIV-1 as chronic infection model with rhesus macaque taint with SIV mac251
Dye has important reference significance.In conventional research, using Zinc finger nuclease (ZFN) or transcriptional activators sample effector core
Sour enzyme (TALEN) carries out the special sex modification of gene to the embryonic cell of macaque, for obtaining transgenosis monkey, but for differentiation
Periphery primary cell genetic modification, the technological means to be used is limited.By the use of adenovirus or adeno-associated virus as
Carrier transmission ZFN or TALEN can human peripheral blood cell carry out appropriate modification, but the utilization of both technologies is more multiple
It is miscellaneous, and adenovirus has immunogenicity to rhesus macaque in itself, may influence the effect of genetic modification.
Therefore SIV slow virus carriers are used, CRISPR/Cas9 is transmitted, the efficiency of infection primary cell can be improved, while
Also more convenient efficiently genes of interest can be modified, improves the security of genetic modification.
The content of the invention
For current CRISPR/Cas9 recombined lentivirus vectors, because people is the natural host of HIV-1, it is with HIV-1
The slow virus of carrier has potential potential safety hazard to people, and to non-human primates primary cell especially T cell efficiency of infection
Very low, the present invention provides slow virus carrier and the application of a kind of sgRNA and its structure, by the dual-gene modifications of CXCR4 and CCR5
Monkey T cell SIV virus infection tests in vitro in show better than wild-type cell antiviral infection ability.
It is that, up to this purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of sgRNA, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1-3.
The nucleotide sequence is as follows：
SEQ ID NO.1：CTACAGCAGTGTCCTCATCCTGG；
SEQ ID NO.2：CAATGTGTCAACTCTTGACAGGG；
In the present invention, the sgRNA can need to only knock out CXCR4 selections SEQ according to being selected the need for knocking out gene
SgRNA shown in ID NO.1, need to only knock out the sgRNA shown in CCR5 selection SEQ ID NO.2, and CXCR4 is if desired knocked out simultaneously
And CCR5, SEQ ID NO.1 and SEQ ID NO.2 can be connected with promoter respectively, import jointly in same plasmid, realize
Knock out simultaneously.
Preferably, the promoter of the sgRNA is U6 promoters.
In the present invention, selection SIVmac251 infection Chinese rhesus monkeys, virus infection chronic phase collection suffer from monkey from
Body cell, including candidate stem cell (HSC), peripheral blood CD4+T cells and external Short-term Culture amplification, build and express Cas9 simultaneously
The slow virus carrier of the sgRNAs of nuclease and targeting CXCR4 and/or CCR5 genes.Because people is the natural host of HIV-1,
Slow virus with HIV-1 as carrier has potential potential safety hazard to people, and to non-human primates primary cell especially T cell
Efficiency of infection is very low, therefore selection is based on SIV SIVmac1A11The 3rd generation slow virus skeleton, it is final to obtain
Slow virus is named as SIV-CRISPR/Cas9, by SIV-CRISPR/Cas9 slow virus Infection in Vitro primary cells, then by base
Because the cell modified is fed back to the monkey that suffers from processed by conditionity, observation and treatment effect.
Second aspect, the present invention provides a kind of SIV-CRISP/cas9 slow virus carriers, and the slow virus carrier is comprising such as
The nucleotide sequence of the sgRNA described in first aspect.
The third aspect, the present invention provides a kind of structure of SIV-CRISP/Spcas9 slow virus carriers as described in second aspect
Construction method, comprises the following steps：
(1) HpaI and ClaI double digestion pCL20cSLFR MSCV-GFP carriers are used, the carrier after digestion is obtained；
(2) PCR amplifications U6 promoter-sgRNA-EFS promoter-Cas9 sequences, are cloned into by way of end recombinates
On carrier after step (1) digestion, the SIV-CRISP/Spcas9 slow virus carriers are obtained.
Preferably, the nucleotide sequence of the primer of the PCR amplifications described in step (2) is as shown in SEQ ID NO.3-4.
The nucleotide sequence is as follows：
SEQ ID NO.3：CGGTGGTTCGAACGCGTTAACCCTATTTCCCATGATTCCTTC；
SEQ ID NO.4：GGTACCGTATACGGCATCGATGTAATCCAGAGGTTGATTGTCG.
Fourth aspect, the present invention provides a kind of recombinant slow virus, by comprising the SIV-CRISP/ as described in second aspect
Cas9 slow virus carriers and packaging helper plasmid pCAG4-RTR-SIV, pCAG-SIVgprre and pCAG-VSVG cotransfection lactation
The recombinant slow virus that cell is obtained.
Preferably, the mammalian cell is HEK293T cells.
5th aspect, the present invention provides a kind of SIV-CRISP/cas9 slow virus carriers as described in second aspect to be used to strike
Except CXCR4 and/or CCR5 genes.
6th aspect, the present invention provides a kind of composition, and the composition includes that the slow virus as described in second aspect carries
Body and/or the recombinant slow virus as described in fourth aspect.
7th aspect, the present invention provides the slow virus carrier as described in second aspect, the recombinant lentiviral as described in fourth aspect
The application of virus or the composition as described in terms of the 6th in anti-AIDS drug is prepared.
Compared with prior art, the present invention has the advantages that：
(1) SIV-CRISPR/Cas9 slow virus of the invention is different from other Cas9 tool for transmitting reported at present, institute
Stating SIV slow virus carriers can successfully be transferred to rhesus macaque primary cell by CRISPR/Cas9 systems, so as to realize to target gene
Effectively knock out, the monkey HSC cells modified by CCR5 still maintain the potential of differentiation, by the dual-gene modifications of CXCR4 and CCR5
Monkey T cell SIV virus infection tests in vitro in show better than wild-type cell antiviral infection ability；
(2) SIV slow virus carriers of the invention have infection ability higher to non-human primate cells, can apply to
The Pre-clinical animal studies of various disease genes treatment, because SIV slow virus will not infect human body, and cell to people source also has
There is efficiency of infection higher, the clinical research it can be considered to be directly used in future；
(3) this patent employs current most efficient CRISPR/Cas9 gene editings instrument, designed CXCR4/CCR5
Gene knockout activity of the gene sgRNA sites with other sites better than conventional research institute report is also first Application in SIV
The gene therapy of rhesus macaque is infected, there is the advantage such as simple operation, with low cost compared to ZFN and TALEN.
Brief description of the drawings
Fig. 1 (A) is the SIV-CRISPR/Cas9 Lentiviral schematic diagrames of present invention targeting CXCR4, and Fig. 1 (B) is
The SIV-CRISPR/Cas9 Lentiviral schematic diagrames of present invention targeting CCR5；
Fig. 2 (A) is the gel electrophoresis spectrum of the optimal CXCR4 specificity sgRNA of present invention screening, and Fig. 2 (B) is the present invention
The gel electrophoresis spectrum of the optimal CXCR4 specificity sgRNA of screening；
Fig. 3 (A) is the detection of GFP expressions, Fig. 3 (B) after monkey CD4+T cell infections SIV-GFP reporter virus of the present invention
It is GFP expressions detection after monkey HSC cell infections SIV-GFP reporter virus of the present invention；
Fig. 4 effectively knocks out monkey CD4+T cell CXCR4 and CCR5 genes for CRISPR/Cas9 slow virus of the present invention, wherein,
Fig. 4 (A) is the gel electrophoresis spectrum after knocking out, and Fig. 4 (B) is the knockout efficiency of CXCR4 genes, and Fig. 4 (C) strikes for CCR5 genes
Except efficiency；
Fig. 5 is that the monkey CD4+T cells of CRISPR/Cas9 modifieds of the present invention can resist the viral infection of SIV；
Fig. 6 effectively knocks out monkey HSC cell CCR5 genes for CRISPR/Cas9 slow virus of the present invention；
Fig. 7 is that the monkey HSC cells of CRISPR/Cas9 modifieds of the present invention still retain the potentiality of hematopoietic differentiation.
Specific embodiment
Further to illustrate technological means and its effect that the present invention is taken, below in conjunction with accompanying drawing and by specific real
Mode is applied to further illustrate technical scheme, but the present invention is not limited in scope of embodiments.
Embodiment 1：Target the SIV-CRISPR/Cas9 Lentivirals pCL20-CAS- of people/monkey CXCR4/CCR5
The structure of sgRNA
(1) from GenBank database search people and the CXCR4 genes and CCR5 genes of rhesus macaque, by sequence alignment, look for
To the homologous sequence of both protein-coding regions.Selection is close to the homologous sequences at 5 ' ends, with online tool (http://
Crispr.mit.edu/) design possible sgRNA, the online tool can according to the base distribution situation of target sequence, with reference to
The possible probability that misses the target, obtains comprehensive grading.Score value sgRNA higher, its is possible, and to knock out efficiency higher and miss the target efficiency more
It is low.Based on this, 7 sgRNA that are in the top and being held near code area 5 ' are chosen in this patent to CXCR4；CCR5 is chosen and is arranged
Name is forward and near 8 sgRNA at the end of code area 5 ', shown in such as Fig. 1 (A)-Fig. 1 (B).
The all sgRNA carriers that will be built transfect Hela-CD4 cells, extract DNA, used for expanding target sequence
Primer mark is：
SEQ ID No.5:CACTTCAGATAACTACACCGAGG；
SEQ ID No.6:GAGTGTGACAGCTTGGAGATG；
SEQ ID No.7:CGCTCTACTCACTGGTGTTC；
SEQ ID No.8:CCTGTGCCTCTTCTTCTCATT；
The PCR primer that will be expanded is used for Surveyor digestions, and subsequent electroresis appraisal goes out has knockout activity higher
SgRNA, as a result as shown in Fig. 2 (A)-Fig. 2 (B), CXCR4 and CCR5 genes have different genes under different sgRNA effects
Knock out efficiency.CRISPR/Cas9 causes target sequence that DNA double chain fracture occurs, and mammalian cell is connected by nonhomologous end
Connecing (NHEJ) repair mode carries out the reparation of chain rupture, and various mutation (InDels) are produced therewith.Surveyor enzymes are then special using
In detection, these are mutated.
(2) selected optimal sgRNA carriers are expanded through PCR, obtains one section of complete sequence comprising sgRNA and Cas9：
U6 promoter-sgRNA-EFS promoter-Cas9, for expand primer mark be：
SEQ ID NO.3：CGGTGGTTCGAACGCGTTAACCCTATTTCCCATGATTCCTTC；
SEQ ID NO.4：GGTACCGTATACGGCATCGATGTAATCCAGAGGTTGATTGTCG.
The sequence that will be expanded is recombinated with the pCL20cSLFR MSCV-GFP long segments of HpaI+ClaI double digestions by end
Mode connect and obtain pCL20-CAS-sgRNA carriers.This step is carried out using the T-A clone kit of Vazyme companies
Operation.
(3) sequencing of recombinant plasmid.Recombinant plasmid is served into Hai Ying fine horses Bioisystech Co., Ltd to be sequenced, sequencing knot
Fruit compares with expected result, it was demonstrated that sequence is completely correct.
Embodiment 2：A large amount of extractions of Lentiviral and package carrier
(1) pCL20-CAS-sgRNA is taken respectively, and pCAG4-RTR-SIV, pCAG-SIVgprre, pCAG-VSVG plasmid are few
Amount conversion stbl3 E. coli competents, second day picking monoclonal is inoculated in LBs of the 1ml containing ampicillin (Amp) and cultivates
Liquid, after shaking bacterium 8 hours, take 500 μ l bacterium solutions access the blake bottle containing 500ml LB/Amp nutrient solutions in, 37 DEG C of shaking table cultures
12-16 hours；
(2) 5000*g is centrifuged 5 minutes, and collects thalline, outwells culture medium and left-hand thread gently pats residual to exhaust in blotting paper
Liquid.The thalline of collection is using the big upgrading grain of the big extraction reagent kit of plasmid (Guangzhou Mei Ji bio tech ltd).
(3) adding 20ml BufferP1/RNase A (ensures that bacterium is completely resuspended, weight to the resuspended bacterium that in thalline, is vortexed
Solution after outstanding does not have cell mass)；
(4) to 20ml Buffer P2 are added in re-suspension liquid, overturn and mix 10-15 times, standing 1-2 minutes (must be light
It is light to mix, note the operating time no more than 4 minutes)；
(5) in lysate add 20ml Buffer E3, immediately overturn mix 15-20 time, blending process will softly and
Fully.Neutralizing sufficient standard is：Solution becomes less viscous thick, sediment dispersion whitening color；
(6) 3000*g is centrifuged 10 minutes, then shifts in supernatant to new centrifuge tube, adds 1/3 times of volume Buffer
E4 is vortexed and mixes in supernatant；
(7) MaxPure Micro Column are enclosed within 50ml centrifuge tubes, in transfer 15ml mixed liquors to pillar,
3000*g is centrifuged 10 minutes；
(8) outwell efflux, during pillar is recovered collecting pipe, in continuing to shift remaining solution to pillar, 3000*g from
The heart 10 minutes；
(9) efflux is outwelled, during pillar is recovered collecting pipe, 20ml Buffer E5 to pillar, 3000*g centrifugations is added
10 minutes；
(10) outwell during pillar recovered collecting pipe by filtrate.Add 20ml Buffer PW2 (being diluted with absolute ethyl alcohol)
To pillar, 3000*g is centrifuged 10 minutes；
(11) outwell during pearl recovered collecting pipe by filtrate.Add 20ml Buffer PW2 (being diluted with absolute ethyl alcohol)
To pillar, 3000*g is centrifuged 10 minutes；
(12) fall to abandon during pillar recovered collecting pipe by filtrate, 4000*g is centrifuged 10 minutes；
(13) pillar is enclosed within the 50ml centrifuge tubes of sterilizing, adds 30-200ml aqua sterilisas to the film center of pillar, it is quiet
Put 2 minutes, 10000*g is centrifuged 5 minutes, collect separating liquid；
(14) further extracting removes endotoxin, with aqua sterilisa adjustment plasmid concentration between 0.1g-0.6mg/ml；
(15) 0.1 times of volume Buffer ER1 and 0.1 times of volume Buffer ER2 is added in plasmid solution, overturning mixed
It is even；
(16) 20min is placed on ice, and period, reverse mixing was multiple；
Room temperature is centrifuged 3min under 10000*g speed after (17) 42 DEG C of water-bath 5min；
(18) sample is gently taken out, it is found that bottom forms one layer of solution of red, supernatant is carefully transferred to centrifugation
Guan Zhong, adds in the 0.7 times of isopropanol of volume to supernatant, overturns and mixes repeatedly；
(19) at room temperature, 10min is centrifuged under 10000*g speed, outwells supernatant, add 1 times of 70% ethanol of volume
Into precipitating.It is vortexed and mixes 15-30s；
(20) at room temperature, 3min is centrifuged under 10000*g speed, carefully outwells supernatant；
(21) at room temperature, the drop that 1min is collected on tube wall is centrifuged under 10000*g speed, precipitation should not be drawn onto, in sky
10min is dried in gas；
(22) add appropriate amounts of sterilized water in plasmid, vortex 10-20s is mixed, and is stored at room temperature 30min and is made plasmid fully molten
Solution, period, reverse mixing was multiple；
(23) plasmid concentration is measured on Nanodrop ultraviolet specrophotometers, then plasmid is stored in -20 DEG C.
Embodiment 3：The packaging of slow virus, concentration and titrate.
The packaging of 3.1 slow virus
(1) the pre- plate of cell：The 24h before transfection, taking the 293T cells for covering with carries out pre- plate, and 70% remittance can be reached with second day
It is right to be advisable (pre- plate 5,000,000 in 75mm culture dishes)；
(2) second days：Transfection；
A () observes the growing state of pre- plate cell, be advisable 90% or so with the degree of converging of cell, sucks culture medium, plus
Enter the complete medium (15mL in 75mm culture dishes) of fresh preheating；
By holding chambers such as the PEI needed for transfection, plasmid, Opti-MEM I Reduced Serum Medium before (b) transfection
Temperature balance；
C () prepares the mixed liquor of plasmid.This packs totally 10 ware 293T cells, and each ware cell prepares the μ L of mixed liquor 500,
Therefore mixed liquor cumulative volume need to be prepared for 5mL.Three kinds of plasmids first are added according to various plasmid consumptions, totally 420 μ g (pCL20-CAS-
sgRNA：pCAG4-RTR-SIV：pCAG-SIVgprre：PCAG-VSVG=5:3:3:3) Opti-MEM I Reduced, are added
Serum Medium adjust volume to 2.5mL, and room temperature places 10min after piping and druming is mixed.PEI and Opti-MEM I are taken again
Each 1.25mL of Reduced Serum Medium, room temperature places 10min after mixing.Finally both the above solution is mixed, is mixed
Room temperature places 10min, the as mixed liquor of transfection afterwards；
D () mixed liquor is added dropwise in culture dish, per the μ L of ware 500；
(3) after transfection 6h, every ware culture medium need to be changed into 17mL UltraCLUTURE serum free mediums；
(4) the three to five days：The collection of virus.Transfection 48h-72h, collects culture supernatant, the viral supernatants of collection respectively
It is temporarily stored into 4 DEG C.
3.2 slow virus purify and concentration
The culture supernatant that will be collected uses cross-flow ultrafiltration film (Millipore TFF BiOmax after the membrane filtration with 0.45um
100k milipore filters bag) purified：Small molecule foreign protein in stoste is filtered, medium is replaced into aseptic without Ca2+、Mg2+
The PBS of ion, while reducing cumulative volume to 30-40ml.Ultracentrifugation concentrates slow virus：The rotor 25000rpm of SW-32Ti is (right
It is 106750g to answer centrifugal force), 4 degree are centrifuged 2 hours.After removing supernatant, the PBS with 400ul is resuspended, is stored in -80 DEG C.
The titration of 3.3 slow virus
Pre- bed board Hela cells, 1 × 10 in (1) 6 orifice plate5Cells/well, 37 DEG C of 5%CO2Overnight incubation in incubator
(about 18-20h)；
(2) polybrene mother liquors are prepared：Sterilizing pure water, 8mg/mL, 0.22 μm of filter filtering, packing is stored in -20 DEG C
It is standby；
(3) the DMEM culture mediums containing polybrene are prepared：+ 10%FBS ,+1% is dual anti-, 8 μ g/mL polybrene (trainings
Support the one thousandth of matrix product)；
(4) virus is melted：Viral frozen stock solution is taken out from -80 DEG C, is melted in room temperature, be placed in after thawing and be brought into iuntercellular on ice
It is standby；
(5) virus is diluted：With 10%FBS DMEM (DF-10) complete mediums containing polybrene for preparing by virus
Stoste carries out 10 times of dilutions, obtains 10-1-10-4Viral dilution liquid.Before noticing that drawing virus liquid every time carries out next step dilution
Mix；
(6) old culture medium is discarded, culture mediums of the 1mL without virus is added in the first hole as negative control.Remaining is per hole
The corresponding viral dilution liquid of middle addition 1mL.37 DEG C of culture 18-20h；
(7) the next morning remove containing virus culture medium, add DF-10 (containing 10U/ml DNase I) incubated in 37 DEG C
15min is educated to remove the nucleic acid of residual；Replace with DF-10 culture mediums of the 2mL without polybrene.
(8) continue to cultivate two days later, suck culture medium, then use trypsin digestion cell；
(9) cell is blown out into individual cells, adds appropriate complete medium to terminate pancreatin reaction, 300g 3min centrifugations are received
Collection cell；
(10) cell DNA is extracted, with reference to blood and tissue gene group extracts kit (Qiagen) specification；
(11) bibliography synthesis WPRE primers and probe, are template with the DNA for being carried, and carry out Real time qPCR anti-
Should.Including two independent reactions, reaction one：The WPRE sequences in slow virus (provirus), reaction two are integrated in amplification：In amplification
Identical DNA profiling is used in ginseng gene RNaseP, two reactions, and reaction condition is completely the same：95 DEG C, 1 minute；95 DEG C, 15 seconds, 60
DEG C, 15 seconds, read plate, totally 45 circulations；4 DEG C, 10 seconds；
(15) computing formula：The provirus copy of titre (IU/ml)=(P × N × D × 1000)/V, P=each genome
Number, cell number when N=is transfected, D=slow virus extension rates, V=adds viral dilution liquid to accumulate, and the numerical value of wherein P is needed
Numerical value according to plasmid standard and RNaseP internal references determines.
Embodiment 4：Separation, culture and the infection of rhesus macaque T cell
The separation of 4.1 PBMC cells
(1) take a blood sample 50mL, and (acceleration is set to 3 to 1100g centrifugations 10min, and 1) reduction of speed is set to；
(2) leukocytic cream is taken, 8mL is diluted to 2%FBS；
(3) in absorption 5mL LymphoPrep additions 15mL centrifuge tubes, then dilute blood is carefully added into layering along tube wall
On liquid, both interface is clears are kept；
(4) (acceleration is set to 1 to 1100g centrifugations 20min, and 1) reduction of speed is set to；
(5) linen mononuclearcell is gently suctioned out with pasteur pipet, another is added and has been contained 10mL RF-10's
In centrifuge tube, mix；
(6) 5min is centrifuged with 500g, abandons supernatant；
(7) 10mL RF-10 re-suspended cells, trypan blue is added to count, 300g centrifugation 10min abandon supernatant.
4.2 magnetic bead sorting rhesus macaque CD4+T cells
(1) it is vortexed and mixes magnetic bead, take 25 μ L magnetic beads in test tube, add 1mL Buffer 1 to mix, test tube is placed on magnetic
1min on power frame, removes supernatant.Plus 25 the μ L resuspended magnetic beads of Bffer1 it is standby；
(2) PBMC with Buffer 1 it is resuspended to density be 107Individual/mL；
(3) to the magnetic bead for adding 25 μ L to wash in the PBMC of 1mL, 2-8 DEG C of incubation 20min is placed on Sloped rotating on shaking table；
(4) test tube is placed on 2min on magnet, collects supernatant；
(5) test tube, plus 1mL Buffer 1 are removed, piping and druming is mixed, and is placed on 2min on magnetic bead, collects supernatant, is repeated once；
(6) repeat step (5)；
(7) plus the re-suspended cells of 100 μ L Buffer 2, plus 10 μ L DETCHaBEAD, be incubated at room temperature 45min make cell from
Discharged on magnetic bead；
(8) test tube is placed on 1min on magnetic frame, the supernatant containing cell is transferred in new test tube, plus 500 μ L
The washings of Buffer 2 magnetic bead 2-3 times, collects supernatant；
(9) add 4mL Buffer 2,350g centrifugation 5min, supernatant is removed, with T cell culture medium re-suspended cell；
The culture of 4.3 rhesus macaque CD4+T cells
(1) the CD4+T cells 350g centrifugations 10min after magnetic bead sorting；
(2) the resuspended countings of RF-10；
(3) CD4+T cells are inoculated in 48-well plates, cell density is 5 × 105Individual/mL；
(4) CD3/CD28 antibody magnetic beads are added in T cell special culture media.The amount of magnetic bead is added in the ratio 1 with cell:
1 adds；
(5) add rhIL-2, make final concentration of 10ng/ml.
(6) count 2-3 times weekly, record the proliferative conditions of cell.
The slow-virus transfection of 4.4 T cells.
(1) the CD4+T cells 350g centrifugation 10min after magnetic bead sorting, plus the resuspended counting of complete medium；
(2) CD4+T cells are added to be cultivated in 96 orifice plates, cell density is 5 × 105Individual/mL, is 1 × 10 per hole cell number5
It is individual；
(3) magnetic bead is washed, is added and cell 1 in culture dish:The magnetic bead of 1 ratio；
(4) add rhIL-2, make final concentration of 10 μ g/L；
(5) after stimulating 24h, infection cell, plus polybrene, make final concentration of 6 μ g/mL, mix；
(6) liquid is changed after 16-24h.
Fluorescence microscope is taken pictures the situation of virus infection T cell, shown in such as Fig. 3 (A).
The T cell genome after gene knockout is extracted, the gene knockout efficiency of CXCR4 and CCR5 is detected, as a result such as Fig. 4
(A), 4 (B), 4 (C) show the gene knockout efficiency of CXCR4 and CCR5.
T cell after gene knockout infected monkey AIDS virus, resistance situation of the detection to AIDS virus in vitro.Result is such as
Shown in Fig. 5, the T cell after CCR5 gene knockouts has repellence to the simian acquired immunodeficiency syndrome poison of CCR5 preferendums.
Embodiment 5：The separation and culture of monkey candidate stem cell (HSC) cell
(1) mobilization of candidate stem cell is carried out to SIV infected monkeys：Joint injection rhSCF (100 μ g/kg/day) and rhG-
CSF (25 μ g/kg/day), for three days on end；
(2) PMNC after mobilizing is separated with blood cell separator, is separated according to the operation of step 4.1
PBMC；
(3) according to CD34 positive cell magnetic bead sorting kit operating instructions, CD34+HSC is sorted.According to 1 × 106/ml
Concentration HSC be laid on be coated with 25 μ g/cm in advance224 well culture plates of RetroNectin fibronectins, culture medium is
The SFEM stem cell medias (StemCell) of StemSpan II, the cell factor of addition has：SCF, Flt3L, TPO, concentration are
100ng/ml, referred to as HSC complete mediums；
(4) cell pre-stimulation (is served only for knocking out after 2 hours according to MOI=100 addition SIV-sgR5/Cas9 slow virus
CCR5), fresh complete medium is replaced with after infection 24h；Virus of fluorescence microscope taking pictures infects the situation of candidate stem cell
As shown in Fig. 3 (B).
(5) after continuing to cultivate 2 days, cell is collected, for detecting CCR5 gene knockout efficiency, as a result as shown in fig. 6, monkey is made
CCR5 genes in hemocytoblast have the knockout of certain efficiency；Evaluate the phenotype of cell after modifying；HSC after detection modification is thin
Ability of the export-oriented multiple hematopoietic lineage differentiation of cell space etc..
Embodiment 6：The Hematopoiesis in Vitro of monkey HSC cells breaks up colony (CFU) analysis
(1) collect and infected SIV-sgR5/Cas9 and the control HSC cells being uninfected by, accurate counting；
(2) each treatment group takes 1000 living cells respectively, is resuspended in 300 μ L HSC complete mediums, supplement addition
G-CSF, IL-3, IL-6, each 50ng/ml；
(3) resuspended cell is transferred in the methylcellulose semisolid culturemedium (StemCell) of 3ml preheatings, concussion is mixed
After even, the 6- orifice plates of low absorption are laid on.Each plate is only inoculated with 1 hole cell, and remaining 5 hole adds each 2ml of sterilized water；
(4) orifice plate is placed in 37 DEG C, is cultivated 12-14 days, observe and count a variety of colonies：BFU-E/CFU-E/
CFU-G/CFU-M/CFU-GM/CFU-GEMN etc., as a result as shown in fig. 7, candidate stem cell after gene knockout has is divided into
The ability of various cell colonies；
(5) all of monoclonal cell colony of picking, for extracting DNA, detects the gene knockout situation of each clone, enters
Row statistics.
Applicant states that the present invention illustrates method detailed of the invention by above-described embodiment, but the present invention not office
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Art
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosing.
SEQUENCE LISTING
<110>Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health
<120>The slow virus carrier and application of a kind of sgRNA and its structure
<130> 2017
<160> 8
<170> PatentIn version 3.3
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Claims (10)
1. a kind of sgRNA, it is characterised in that the nucleotide sequence of the sgRNA is as shown in SEQ ID NO.1-2.
2. sgRNA according to claim 1, it is characterised in that the promoter of the sgRNA is U6 promoters.
3. a kind of SIV-CRISP/cas9 slow virus carriers, it is characterised in that the slow virus carrier comprising such as claim 1 or
The nucleotide sequence of the sgRNA described in 2.
4. a kind of construction method of SIV-CRISP/Spcas9 slow virus carriers as claimed in claim 3, it is characterised in that bag
Include following steps：
(1) HpaI and ClaI double digestion pCL20cSLFR MSCV-GFP carriers are used, the carrier after digestion is obtained；
(2) PCR amplifications U6 promoter-sgRNA-EFS promoter-Cas9 sequences, step is cloned into by way of end recombinates
(1) on the carrier after digestion, the SIV-CRISP/Spcas9 slow virus carriers are obtained.
5. method according to claim 4, the nucleotide sequence such as SEQ ID of the primer of the PCR amplifications described in step (2)
Shown in NO.3-4.
6. a kind of recombinant slow virus, it is characterised in that will be carried comprising SIV-CRISP/cas9 slow virus as claimed in claim 3
The weight that body is obtained with packaging helper plasmid pCAG4-RTR-SIV, pCAG-SIVgprre and pCAG-VSVG cotransfection mammalian cell
Group slow virus.
7. recombinant slow virus according to claim 6, it is characterised in that the mammalian cell is HEK293T cells.
8. a kind of SIV-CRISP/cas9 slow virus carriers as claimed in claim 3 are used to knock out CXCR4 and/or CCR5 bases
Cause.
9. a kind of composition, it is characterised in that the composition includes slow virus carrier as claimed in claim 3 and/or such as
Recombinant slow virus described in claim 6.
10. slow virus carrier as claimed in claim 3, recombinant slow virus as claimed in claim 6 or such as claim 9 institute
Application of the composition stated in anti-AIDS drug is prepared.
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| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN110055281A (en) * | 2019-04-25 | 2019-07-26 | 山东大学第二医院 | A kind of slow virus carrier being used to prepare CAR-T and its construction method and application |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
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