CN107475255A - A kind of sgRNA based on CRISPR/Cas9 gene editing systems of genophore mediation and application thereof - Google Patents

A kind of sgRNA based on CRISPR/Cas9 gene editing systems of genophore mediation and application thereof Download PDF

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CN107475255A
CN107475255A CN201710167563.3A CN201710167563A CN107475255A CN 107475255 A CN107475255 A CN 107475255A CN 201710167563 A CN201710167563 A CN 201710167563A CN 107475255 A CN107475255 A CN 107475255A
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sacas9
sod1
sgrna
aav9
nucleotide sequence
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CN107475255B (en
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段伟松
吴小兵
李春岩
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Second Hospital of Hebei Medical University
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BEIJING FIVEPLUS MOLECULAR MEDICINE INSTITUTE CO LTD
Beijing Ruixi Rare Disease Gene Treatment Technology Institute
Second Hospital of Hebei Medical University
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Abstract

The present invention provides one kind genophore（Including but not limited to 9 type adeno-associated viruses）Mediation, based on CRISPR/Cas9 gene editing system direct editings superoxide dismutase 1（Superoxide Dismutase 1, abbreviation SOD1）Mutator, amyotrophic lateral sclerosis is treated in vitro and in transgenic mice（Amyotrophic Lateral Sclerosis, abbreviation ALS）Gene therapy method.Recombinant virus provided by the invention has biological activity, promises to be the viral candidates of als gene treatment.

Description

A kind of sgRNA based on CRISPR/Cas9 gene editing systems of genophore mediation And application thereof
Technical field
The present invention relates to biological technical field, more particularly to a kind of AAV9 viral vectors mediation based on CRISPR/Cas9 SgRNA of gene editing system and application thereof, include the composition with the sgRNA, recombinant expression carrier and gene therapy mode.
Background technology
ALS（Amyotrophic Lateral Sclerosis, ALS）It is a kind of nerve of lethal Degenerative disease, a type as motor neuron disease（ALS/MND）, be most common adult onset motor neuron Disease.ALS Ahl tribulus sea silent sickness, Parkinson's are all nervous system common degenerative disease, but development speed and property are compared with it His two kinds of degenerative diseases are more serious.Using spinal cord, brain stem, corticocerebral motor neuron degenerative change occurs for patient as spy Sign, shows as progressive muscle weakness, dies from respiratory failure more；Adult onset, median survival interval are only 3-5 [1].It is ill Rate 4-6/10 ten thousand, incidence of disease 1-3/10 ten thousand.So far it there is no effective treatment method [2].Now clinically it is used for the medicine for treating ALS " Rilutek " is only capable of delaying the course of disease several months.Neurologist, nerve specialist ALS more is referred to as " cancer for not being cancer ".In being apt to occur in due to this disease Year crowd, i.e. the main support crowd of society and family, therefore white elephant is brought to family, national and society is caused Serious harm.By establishing effective remedy measures, the pain of patient can not only be mitigated, and can give family, country and Society reduces the heavy burdens.
Patient is common to distribute by ALS, accounts for the 90% of total patient, familial accounts for 10%.Gene mutation is that ALS identifications are clear and definite The cause of disease.Familial amyotrophic lateral sclerosis more than 50%（FALS）The related genes of patient have been found that and wherein wrapped Include：C9orf72 (24%), SOD1 (20%), TDP-43 (5%), FUS (5%) etc..Sporadic ALS（SALS） Patient includes：[3] such as C9orf72 (4%), SOD1 (2%), TDP-43 (1%), FUS (1%).Base is carried out to mutator Because of editor, repair mutator and be possible to fundamentally retardation motion neuronal degeneration.
The short palindrome repetitive sequence-CRISPR associated protein 9s of the regular intervals of cluster（CRISPR-Cas9）System is nearest That establishes can apply in eukaryotic, the gene editing system by RNA regulation and control to DNA modification.With other editing systems Compare, such as Zinc finger nuclease（ZFNs）With class activating transcription factor sample effector nuclease（TALENs）, CRISPR-Cas9 systems Editorial efficiency and targeting are higher, and it is easy to practice shooting, and have the characteristics that more site modifications [4,5].Cas9 nucleases are sgRNA's The Yuan Jian areas adjacent sequences of the lower mediation target gene of guiding（PAM sequences, NGG）The double-strand break of 3 bases in upstream, on Cas9 HNH domains cut the single stranded DNA that complementation is formed with sgRNA, and Ruv C-structure domain cuts the single-stranded of incomplementarity, passes through endogenous DNA is repaired, and carries out non-homologous end joining（nonhomologous end joining, NHEJ）, form insertion or missing be prominent Become（indel mutation）, complete gene knockout；Under the conditions of existing for template strand, homologous recombination repair is completed （Homology-Directed Repair, HDR）[6-8].
Adeno-associated virus（Adeno-associated virus, AAV）The carrier safety barrier new as one kind, its Importance is just gradually recognized by researcher.It is parvovirus family member, is nonencapsulated linear ssdna virus, has There is extensive host range, somatoblast and can infection Unseparated Cell, and the long-term table of energy mediate foreign gene can be infected Reach.As important a member of viral vector, AAV does not have obvious cytotoxic effect, and will not be as other viral vectors Cause the strong immune response of cell like that；Simultaneously structure recombinate AAV viruses when, can make its coded sequence completely missing and only Retain its 145 bp terminal repeat, so as to effectively reduce the possibility of its restructuring and expression oneself protein, make security It is further enhanced.Therefore, AAV is intriguing more and more as a kind of preferable gene therapy vector With concern [9,10].
Chengzu Long etc. are in Du Shi muscular dystrophy（Duchenne Muscular Dystrophy, DMD）Turn base Because utilizing CRISPR-Cas9 systems in mouse, the reparation to mutator in fertilized egg cell is completed, is experimentally observed As long as the gene repair can of newborn mice part cell reaches the complete recovery [11] of phenotype.Lukasz Swiech etc. are built AAV-SpGuide has been found, the AAV-SpCas9 points of systems for opening expression, has passed through 1:1 be blended in hippocampus of mice injection, SpGuide and SpCas9 cotransductions efficiency is 75%, and the SpCas9 of miss rate 0-1.6%, AAV mediation is expressed in neuron not to be shown To the toxicity of neuron.And then author observes, the Dnmt1 allele modification efficiency of AAV mediations>60%, Dnmt3a and Dnmt3b allele modification rates are respectively 42% and 17%；The expression of Dnmt1 protein levels is remarkably decreased, under Dnmt3a albumen Drop has exceeded 50%, and Dnmt3b albumen is nearly no detectable.These results prompting CRISPR-Cas9 systems can be by non-same The gene of knockout mutations is repaired in the connection of source end, reaches the effect [12] for the treatment of.Recently, open peak etc. realize sgRNA and SaCas9 is expressed in an AAV carrier, and saCas9 encoding base number is small compared with spCas9 by 25%, further increases gene volume The efficiency [13,14] collected.
ALS caused by SOD1 gene mutations is 20% in familial ALS, is 2% in sporadic ALS. The mechanism that SOD1 mutation cause motor nerve degeneration is unclear, may be with oxidative stress, er stress, mitochondrial function Obstacle, proteasome/autophagy path is abnormal, dystrophia, and activation of spongiocyte etc. is relevant.But the nerve of Mutation SOD1 Toxicity is a kind of acquired toxicity, and function loss is unrelated [15] after being mutated with SOD1.Therefore, CRISPR-Cas9 systems pair are utilized Mutation SOD1 gene carries out gene editing, one section of sequence of modification SOD 1 on DNA level, by endogenous dna reparation, knocks out Mutation SOD1 gene, remove the acquired toxicity of Mutation SOD1.
SOD1-G93A semizygote transgenic mices are research neuromuscular disorder diseases, for example ALS or Ge Lei kirschner disease is very Good animal model.The G93A mutation of the transgenic mice high copy number expression gene of hSOD 1, thus have due to spinal motor Neure damage and the one or more limb paralysises paralysis occurred, show the phenotype similar to people ALS.Therefore SOD1G93A Gene expression pattern can turn into the ALS animal models of unique label.
A load that can target motor neuron is also needed to mutator editor on Mutation SOD1 transgenic mice System is united.Adeno-associated virus 9 is a kind of hypotoxicity, low immunogenicity, and the one kind that can apply to clinic is inverse by aixs cylinder The vector virus [16-18] of row transport targeting motor neuron.2012, European drugs administration approved was by adeno-associated virus Active lpl gene is inserted in myocyte and treats lipoprotein lipase deficiency.In nearest clinical trial, it was confirmed that gland Related viral vectors can carry therapeutic gene to there is no the diseases for the treatment of means, such as hemophilia, choroideremia disease etc., energy at present The effect of reaching healing or control progression of disease [19,20].
Therefore, the AAV9 carrier systems for CRISPR-Cas9 systems being combined to targeting motor neuron transport are dashed forward in body editor The SOD1 genes of change, which are one, can fundamentally solve the method that SOD1 mutation cause motor nerve degeneration.
The content of the invention
In view of this, the present invention provide a kind of mediation of AAV9 viral vectors based on CRISPR/Cas9 gene editing systems SgRNA and application thereof, the composition comprising the sgRNA, recombinant expression carrier and gene therapy mode.The sgRNA can be effective The SOD1 genes of mutation are edited, repair the motor neuron of denaturation, so as to reach treatment ALS purpose.It is provided by the invention heavy Group virus has biological activity, promises to be the viral candidates of als gene treatment.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of sgRNA, it is characterised in that it has：
（Ⅰ）, nucleotide sequence as shown in SEQ ID No.1；Or
（Ⅱ）, nucleotide sequence as shown in SEQ ID No.1 complementary series；Or
（Ⅲ）And（Ⅰ）Or（Ⅱ）Nucleotide sequence coded same protein, but because of the degeneracy of genetic code and with（Ⅰ）Or （Ⅱ）The different sequence of nucleotide sequence；Or
（Ⅳ）And（Ⅰ）Or（Ⅱ）Or（Ⅲ）The sequence of the sequence at least 70% homology.
It is used for the purposes for editing SOD1 genes present invention also offers the sgRNA.
Present invention also offers a kind of composition, including AAV9 and SaCas9 and described sgRNA.
Present invention also offers the preparation method of the composition：
Step 1：Albumen needed for coding AAV9 duplications is connected with coding SaCas9 albumen with described sgRNA nucleotide sequence Into carrier, construction of expression vector；
Step 2：The expression vector is converted into host cell, expression, expression product is collected, produces.
Present invention also offers a kind of recombinant vector expression unit, including：
（1）Encode the nucleotide sequence of albumen needed for AAV9 duplications；And/or
（2）Encode the nucleotide sequence of SaCas9 albumen；And/or
（3）Encode sgRNA as claimed in claim 1 DNA.
In some specific embodiments of the present invention, the construction method of the recombinant vector expression unit, it will encode The nucleotide sequence of albumen and coding SaCas9 albumen and the sgRNA is connected in carrier needed for AAV9 duplications, construction expression Carrier.
In some specific embodiments of the present invention, the carrier in the construction method of the recombinant expression carrier is plasmid Or virus.
In some specific embodiments of the present invention, virus is included but not in the construction method of the recombinant expression carrier It is limited to adeno-associated virus, slow virus, adenovirus, herpes simplex virus, rabies viruses and related derivatives.
The structure of the recombinant vector expression unit is ITR-CMV promoter-SaCas9-BGH poly (A) Signal-U6 promoter-SOD1 sgRNA-ITR AAV9 recombinant expression carriers, abbreviation AAV9-SaCas9-sg1/5.
The recombinant vector expression unit includes：ITR (adeno-associated virus 2 inverted terminal repeat sequence)、CMV promoter (the cytomegalovirus promoter)、SaCas9 (Staphylococcus aureus Cas9), BGH (Bostaurus growth hormone), polyA (polyadenosines Acid), U6 promoter, SOD1 sgRNA (SOD1 small guide RNA) and ITR (adeno-associated virus 2 inverted terminal repeat sequence)。
Wherein, ITR sequence (Patent WO0220748) sequence is as shown in SEQ ID No.2；CMV Promoter sequence is as shown in SEQ ID No.3；SaCas9 sequence is as shown in SEQ ID No.4；BGH poly(A) Signal sequence is as shown in SEQ ID No.5；U6 promoter sequence is as shown in SEQ ID No.6；SOD1 sgRNA's Sequence is as shown in SEQ ID No.1.
Present invention also offers the application of the composition, the recombinant expression carrier in the medicine for preparing treatment ALS.
In some specific embodiments of the present invention, the ALS is nerve degenerative diseases.
The experiment proves that designed SOD1 sgRNA can effectively mediate SaCas9 to carry out base to it in vitro Because of editor（Fig. 1）.In SOD1 cDNA 5 sgRNA of G93A mutational sites upstream engineer（Figure 1A）, according to frameshift mutation, SOD1G93A mutational sites can be cut away under RNA leading by SaCas9.With luciferase reporter gene system to sgRNA Cutting efficiency detected.SOD1 cDNA is inserted among luciferase gene and destroys its coding, if Cas9 mediations Gene editing can cut away SOD1 cDNA, then have an expression of luciferase, therefore can be by detecting the work of luciferase Property determines sgRNA editorial efficiency.It is thin that luciferase reporter gene carrier and SaCas9-sgRNAs carrier cotransfections are entered into 293T In born of the same parents, it is different degrees of that testing result shows that each sgRNA plays the role of, and the more other sgRNAs of sgRNA1 and sgRNA5 have more High efficiency（Figure 1B）.Transfect SaCas9-LacZ into stable expression SOD1-GFP cell line, SaCas9-sgRNA1 or After SaCas9-sgRNA5 DNA, 48h with Flow cytometry GFP fluorescence intensity with determine sgRNA to SOD1 genes compile The mediation efficiency collected.Compared with control group sgLacZ, after transfection SOD1 sgRNA1 or sgRNA5, under fluorescence intensity is obvious Drop（Fig. 1 C）.The protein expression level of SOD1 in Transfected cells is detected with Western blotting, and control group sgLacZ Compare, after the sgRNA1 or sgRNA5 that transfect SOD1, SOD1 protein levels are remarkably decreased（Fig. 1 D）, show designed sgRNA1 and SgRNA5 can effectively mediate SaCas9 to carry out gene editing to SOD1 in vitro.With adeno-associated virus, slow virus, herpe simplex Virus, rabies viruses and several different carriers of nano material carry sgRNA5 and enter 293T cells, and sgRNA5 is in cell for detection Interior activity, as a result show that each carrier is respectively provided with different degrees of delivering effect, wherein the effect with gland relevant viral vector It is most notable（Fig. 1 E）.
AAV virus packagings are further carried out to the recombinant vector using three plasmid co-transfection methods, are purified, concentration, Real- Time PCR determine its final titre.Choosing has the 9 type AAV viruses of more preferable compatibility to neuronal cell.With intracerebroventricular Mode injects 3*10 respectively into newborn SOD1G93A transgenic mices13Vg/ml AAV9-SaCas9-LacZ, AAV9- SaCas9-sgRNA1, AAV9-SaCas9-sgRNA5 or AAV9-SaCas9-sgRNA1+sgRNA5 virus, it can be seen that and it is right Compared according to a group AAV9-SaCas9-LacZ, AAV9-SaCas9-sgRNA1, AAV9-SaCas9-sgRNA5 and AAV9-SaCas9- Obvious postpone is presented in the morbidity of sgRNA1+sgRNA5 group SOD1G93A transgenic mices（LacZ: 105±5.2d (n=5), sg1:122±9.7 d (n=5), sg5: 149±9d (n=5), sg1+sg5: 125±6.6d (n=5), *P < 0.05）, it is especially the most obvious with AAV9-SaCas9-sgRNA5 groups（Fig. 2A and Fig. 5）.The survival rate for the treatment of group's transgenic mice Also it is obviously prolonged compared with control group（LacZ: 132±1.1d (n=5), sg1:143±11.6d (n=5), sg5: 161± 6.8d (n=5), sg1+sg5: 137±6.2d (n=5), *P <0.05）, especially with AAV9-SaCas9-sgRNA5 groups most To be obvious（Fig. 2 B and Fig. 5）.Show that AAV9-SaCas9-sgRNAs can effectively prevent motor nerve degeneration in vivo.
Further mice behavior ability is tested to assess therapeutic effects of the SOD1 sgRNAs to ALS.117 It when to transgenic mice carry out back leg extension test, tested respectively with footprinting at 125 days the 115th day.AAV9-SaCas9- LacZ control group mices are after seizure of disease, and presentation is trembled, walk imbalance, hind leg muscle atrophy, Body weight loss and expiration Expiratory dyspnea, and rapid deterioration within 4 weeks.Compared with control group, AAV9-SaCas9-sgRNA1 and AAV9-SaCas9- The back leg stretched width of sgRNA5 group mouse significantly improves（LacZ: 1.58±0.99 cm (n=5), sg1: 6.09±0.75 cm (n=4), sg5: 11.74±0.75 cm (n=5), sg1+sg5: 4.74±1.26 cm (n=5), *P <0.05） （Fig. 3 A）.Footprinting surveys AAV9-SaCas9-LacZ, AAV9-SaCas9-sgRNA1, AAV9-SaCas9-sgRNA5 and AAV9- Longest distance between the hind leg footprint of SaCas9-sgRNA1+sgRNA5 each groups mouse two, it was LacZ respectively at the 115th day: 3.18±0.7 cm (n=5), sg1: 5.7±0.32 cm (n=4), sg5:5.98±0.29 cm (n=5), sg1+ sg5:4.16 ± 1.22 cm (n=5), it was LacZ respectively at the 125th day: 2.1±1 cm (n=5), sg1: 5.23± 1.2 cm (n=4), sg5:6.4 ± 0.53 cm (n=5), and control group are all significantly improved than treatment group Behavioral feature （Fig. 3 B）, show that intracerebroventricular AAV9 SOD1 sgRNAs, especially AAV9-SaCas9-sgRNA5 can be effectively improved ALS and turn base Because of the exercise performance of mouse.
Choose the most significant AAV9-SaCas9-sgRNA5 of therapeutic effect, further with fluorescence microscope its to motion The protective effect of neuron.Motor neuron and microglia cell are dyed by the use of Neu N and IBA1 as mark Mark, it can be seen that more than 20 μm, cell has been denatured motor neuron cell diameter positive control group Neu N, and AAV9-SaCas9-sgRNA5 groups then remain a considerable amount of proper motion neuronal cells（LacZ: 1.5±0.31 (n= 14), sg5: 4.4±0.28 (n=12), *P <0.05）, microglia cell positive IBA1 is then without this change （Fig. 4 A）.After AAV9-SaCas9-sgRNA5 is treated, the atrophy situation of tibialis anterior is also compared with AAV9-SaCas9-sgLacZ pairs It is obviously improved according to group（LacZ: 1242±667µm2 (n=605), sg5: 1679±665µm2 (n=421), *P < 0.05）（Fig. 4 B）.It is the SOD1 bases mediated by AAV9-SaCas9-sgRNA5 for further protection of the checking to motor neuron Caused by knockout, dyeing observation is carried out to SaCas9 the and SOD1 mutant expression in mouse movement neuronal cell. In AAV9-SaCas9-LacZ control groups, SaCas9 and SOD1 mutant expression is very high, and in AAV9-SaCas9- SgRNA5 treatment groups, the SOD1 mutant expression in SaCas9 positive cells are then very low（LacZ: 2722±210 (n= 10), sg5: 216±52 (n=17), *P <0.05）（Fig. 4 C）, the recombinant virus for showing to obtain has biological activity, Promise to be the viral candidates of als gene treatment.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing There is the required accompanying drawing used in technology description to be briefly described.
Fig. 1 shows the gene editing analyses of SOD1 sgRNAs in vitro；Fig. 1（A）：According to SaCas9 design principles, in SOD1 Five target sgRNA are designed at the G93A mutation of cDNA upstreams；Fig. 1（B）：Detected with luciferase reporter gene system different SgRNAs editorial efficiency, compared with LacZ-sgRNA control groups（mean±SE, n=3, * P < 0.05）；Fig. 1 （C）：Sg-LacZ, SOD1-sgRNA1 and SOD1-sgRNA5 are transfected respectively in stable expression SOD1-GFP 293T cells, are flowed Formula cell art detects GFP expression intensities（sg-LacZ: 3784±678.9, sg1: 3784±32.1, sg5: 1619± 489, mean±SE, n=3, * P < 0.05）；Fig. 1（D）：Sg-LacZ is transfected in Western blot detection 293T cells, SOD1 protein expression level after SOD1-sgRNA1 or SOD1-sgRNA5（sg-LacZ: 0.95±0.06, sg-1: 0.72 ±0.12, sg-5: 0.67±0.14, mean±SE, n=3, * P < 0.05）；Fig. 1（E）：With luciferase reporting base Because system detectio different carriers carry sgRNA5 editorial efficiency, compared with transfection control group（mean±SE, n=3, * P < 0.05, ** P < 0.01, *** P < 0.001）.
Fig. 2 shows the therapeutic effect of the SaCas9-sgRNAs in SOD1G93A transgenic mices；Distinguished in a manner of intracerebroventricular Give SOD1G93A transgenic mices 3*1013Vg/ml AAV9-SaCas9-sgLacZ, AAV9-SaCas9-sg1, AAV9- SaCas9-sg5 or AAV9-SaCas9-sg1+sg5 viruses, mouse invasion rate Fig. 2（A）With survival rate Fig. 2（B）；
Fig. 3 shows that SOD1G93A transgenic mices receive the behaviouristics change after different AAV9-SaCas9-sgRNAs treatments；Fig. 3 （A）：Through AAV9-SaCas9-sgLacZ, AAV9-SaCas9-sg1, AAV9-SaCas9-sg5 and AAV9-SaCas9-sg1+sg5 After treatment, back leg extension test is carried out to transgenic mice at 117 days；Fig. 3（B）：Footprinting detects 115 days and 125 day age The hind leg footprint of mouse two between longest distance（mean±SE, n=4-5, * P < 0.05）；
Fig. 4 shows protective effects of the SOD1 sgRNAs to motor neuron；Fig. 4（A）：SOD1G93A transgenic mices are through AAV9- After SaCas9-sgLacZ or AAV9-SaCas9-sg5 is treated, immuno-fluorescence assay Neu N positive exercises neurons and IBA1 positive microglia cell morphological changes（mean±SE, n=12-14, * P < 0.05）；Fig. 4（B）： SOD1G93A transgenic mices are after AAV9-SaCas9-sgLacZ or AAV9-SaCas9-sg5 treatments, and flesh withers before mouse tibia Contracting situation of change（mean±SD, n=~500, *P <0.05）；Fig. 4（C）：After double dye SaCas9 and SOD1 mutant, fluorescence is common SaCas9 and SOD1 expressions in mouse movement neuron when focusing microscope observation is last eventually（mean±SE, n=10-17, * P < 0.05）；
Fig. 5 shows SOD1G93A transgenic mices after AAV9-SaCas9-sgLacZ or AAV9-SaCas9-sg5 are treated, small Gene copy number, sex, morbidity and the life cycle of mouse.
Embodiment
The invention discloses a kind of sgRNA based on CRISPR/Cas9 gene editing systems of AAV9 viral vectors mediation And application thereof, comprising the composition with the sgRNA, recombinant expression carrier and gene therapy mode, those skilled in the art can be with Present disclosure is used for reference, is suitably modified technological parameter realization.In particular, all similar replacements and change are to ability It is it will be apparent that they are considered as being included in the present invention for field technique personnel.The method and application of the present invention has been led to Preferred embodiment is crossed to be described, related personnel substantially can not depart from present invention, in spirit and scope to this paper institutes The methods and applications stated are modified or suitably changed with combining, to realize and using the technology of the present invention.
First purpose of the present invention is to provide the sgRNA that can effectively mediate SOD1 mutant gene editors.
The different sgRNA are sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, especially sgRNA5.
Second object of the present invention is to provide a kind of carrier, the vector expression AAV9 replicate needed for albumen and SaCas9 albumen.
Third object of the present invention is to provide a kind of AAV9 and SaCas9 albumen and SOD1 sgRNAs recombinant vector tables Up to the preparation method of unit, this recombinant vector expression unit structure is ITR-CMV promoter-SaCas9-BGH poly (A) Signal-U6 promoter-SOD1 sgRNA-ITR AAV9 recombinant expression carriers, abbreviation AAV9-SaCas9-sg1/5.
The recombinant vector expression unit includes：ITR (adeno-associated virus 2 inverted terminal repeat sequence)、CMV promoter (the cytomegalovirus promoter)、SaCas9 (Staphylococcus aureus Cas9)、BGH(Bostaurus growth hormone)、polyA（Polyadenylic acid）、 U6 promoter、SOD1 sgRNA（SOD1 small guide RNA）With ITR (adeno-associated virus 2 inverted terminal repeat sequence)。
Wherein, ITR sequence (Patent WO0220748) sequence is as shown in SEQ ID No.2；CMV Promoter sequence is as shown in SEQ ID No.3；SaCas9 sequence is as shown in SEQ ID No.4；BGH poly(A) Signal sequence is as shown in SEQ ID No.5；U6 promoter sequence is as shown in SEQ ID No.6；SOD1 sgRNA's Sequence is as shown in SEQ ID No.1.
The carrier is plasmid or virus, and the virus includes but is not limited to adeno-associated virus, slow virus, adenovirus, list Pure herpesviral, rabies viruses and related derivatives.
Fourth object of the present invention is to provide a kind of composition, including albumen and SaCas9 albumen needed for AAV9 duplications With the sgRNA of designed SOD1 mutant.
In the composition, the sgRNA of the SOD1 mutant is sgRNA5, and its sequence is as shown in SEQ ID NO.1. SaCas9 sequence is as shown in SEQ ID NO.4.
The 5th purpose of the present invention is in the carrier, the composition or viral in ALS is treated that provide any of the above-described Purposes.
The ALS is nerve degenerative diseases.
In the present invention, using the modern biology such as genetic engineering technology and method, there is provided coding SOD1 sgRNA5, The SaCas9 restructuring AAV9 coexpression vectors and preparation of virus, packaging and its application.
The invention provides sgRNA and application thereof, is controlled comprising the composition, recombinant expression carrier and gene with the sgRNA Treatment mode.Wherein, unless otherwise specified, the various reaction reagents being related in embodiment can be bought by commercial channel Arrive；Unless otherwise specified, the concrete operations being related in embodiment referring to《The Molecular Cloning:A Laboratory guide third edition》.
With reference to embodiment, the present invention is expanded on further：
The AAV9-SaCas9-SOD1 sgRNA of embodiment 1 are cloned and virus packaging
According to SaCas9 PAM sequences（NNGRRT）And the design principle of optimal 21-nt length, design five SOD1 SgRNAs, respectively with after Bsa I digestions, it is cloned into AAV-SaCas9-sgRNA carriers（Addgene, px61591）In.With three Plasmid co-transfection method packaging AAV-SaCas9-SOD1 sgRNAs viruses, purifying concentration, its titre are determined with qPCR.
SOD1 sgRNA are designed and editorial efficiency result：
Fig. 1（A）Show five SOD1 sgRNAs sequences Design；
Fig. 1（B）Show that five SOD1 sgRNA can mediate cuttings of the saCas9 to SOD1 in various degree, wherein with sgRNA1 and SgRNA5 effects are best；
Fig. 1（E）Show that six kinds of carriers have different degrees of delivery efficiency to sgRNA5, wherein best with the effect of adeno-associated virus.
The Flow cytometry SOD1 sgRNA of embodiment 2 gene editing efficiency
SOD1-GFP plasmids are transfected into 293T cells, receive cell after 48h, with 0.25% trypsin digestion cell, centrifuging to sink Form sediment.It is 1 × 10 that cell, which is resuspended, with culture medium7Cells/ml cell suspensions, use flow cytometer（FACSAria, BD）Sorting Go out GFP positive cells, that is, obtain SOD1-GFP stable cell lines.According to the operation manuals of Lipofectamine 2000, to stabilization Express the DNA, 48h that SaCas9-LacZ, SaCas9-sgRNA1 or SaCas9-sgRNA5 are transfected in SOD1-GFP cell line Afterwards with flow cytomery GFP fluorescence intensity to determine mediation efficiency of the sgRNA to SOD1 gene editings.It can be seen that Compared with control group sgLacZ, after transfection SOD1 sgRNA1 or sgRNA5, fluorescence intensity is decreased obviously.
Flow cytometry SOD1 sgRNA gene editing efficiencies：
Fig. 1（C）Show after transfecting SOD1-sgRNA1 and SOD1-sgRNA5 in stable expression SOD1-GFP 293T cells, GFP Expression intensity is decreased obviously.
The Western Blot of embodiment 3 detection SOD1 sgRNA gene editing efficiency
Use total protein extraction kit（Applygen Technologies Inc., P1250）Extract total egg in spinal cord In vain.Each μ g albumen of sample loading 50 carries out PAGE gel electrophoresis after determining concentration, is transferred to pvdf membrane rear enclosed, passes through Beta-actin antibody（1:500, Proteintech）With hSOD1 antibody（1:1, Abcam）After 4oC is incubated overnight, thoroughly wash Wash, the secondary antibody with fluorescence labeling is being incubated at room temperature 1 hour, and then respective strap is carried out using Odyssey infrared imaging systems Detection.It can be seen that SOD1 protein expression level is decreased obviously after SOD1-sgRNA1 or SOD1-sgRNA5 is transfected.
SgRNA1 and sgRNA5 can mediate SaCas9 to carry out gene editing result to SOD1 in vitro：
Fig. 1（D）Show after transfecting SOD1-sgRNA1 or SOD1-sgRNA5 in 293T cells, SOD1 protein expression level is obvious Decline.
The foundation of the mouse model of embodiment 4
SOD1G93A transgenic mices（B6SJL-TgN [SOD1-G93A] 1Gur）Obtained from Jackson laboratories, round the clock The 12h cycles replace, relative humidity 60 ± 10%, are raised under the conditions of 22 ± 1oC of temperature.
The intracerebroventricular AAV of embodiment 5 viruses
AAV is diluted to virus liquids of the 4 μ l containing 0.05% trypan blue, using lambdoidal suture and sagittal suture junction as zero point, forward 1.5mm, at the 0.8-1mm of sagittal suture side, injection depth about 3mm, pin retains 1 minute, then draws back at leisure [21].
The mice behavior of embodiment 6 is analyzed
The motion function of mouse, deadline 180s are tested with turn-club test once in a week, measurement three times, is chosen most long Time records.Disease time is determined according to the decline degree of mouse transfer rod performance weekly.Footprinting is according to the continuous fortune of mouse Move to record, choose the maximum between three step-lengths to be calculated.
It can be seen that giving SOD1G93A transgenic mices 3*10 respectively in a manner of intracerebroventricular13vg/ml AAV9- It is small after SaCas9-sgLacZ, AAV9-SaCas9-sg1, AAV9-SaCas9-sg5 or AAV9-SaCas9-sg1+sg5 virus Obvious postpone is presented in sgRNAs treatment groups in the incidence of disease of mouse, and locomitivity significantly improves.
SOD1G93A transgenic mices receive incidence and behaviouristics after different AAV9-SaCas9-sgRNAs treatments Result of variations：
（1）Obvious postpone is presented in the incidence of disease for the treatment of group in SOD1G93A transgenic mices, sees Fig. 2（A）；
（2）SOD1G93A transgenic mices significantly improve in the survival rate for the treatment of group, see Fig. 2（B）；
（3）Back leg extension test is carried out to transgenic mice at 117 days, the mouse back leg stretched width for the treatment of group significantly improves, See Fig. 3（A）；
（4）Longest distance when footprinting detects 115 days and 125 days between the hind leg footprint of mouse two, treatment group significantly improve, and see figure 3（B）.
The muscle morphology of embodiment 7 is observed
In Olympus microscope after the muscle cross sections of 10 μm of frosts are dyed with HE（BX53）Lower observation coloration result is simultaneously clapped According to image DP73 software analysis.The tibialis anterior of 500 fibers is about recorded, zone of fiber is calculated with software image J.Can To see, after AAV9-SaCas9-sgRNA5 is treated, amyotrophia situation is compared with AAV9-SaCas9-sgLacZ pairs before mouse tibia It is obviously improved according to group.
After AAV9-SaCas9-sgRNA5 is treated, amyotrophia situation detection result before mouse tibia：
Fig. 4（B）Show SOD1G93A transgenic mices after AAV9-SaCas9-sg5 is treated, compared with AAV9-SaCas9-sgLacZ pairs According to group, the tibialis anterior atrophy situation of mouse is obviously improved.
The immuno-fluorescence assay motor neuron cell degenerative condition of embodiment 8
Cell is washed three times with the PBS containing 0.3% Triton X-100, closed 30 minutes in room temperature with 10% horse serum.By phase The dilution proportion answered simultaneously is incubated primary antibody：IBA1 (1:400, Wako, Richmond)、Neu N (1:100, Millipore)、 hSOD1 (1:200, Abcam)、HA (1:300, Sigma) wash three times after, being incubated at room temperature 2h, existed with fluorescence labeling secondary antibody 1 h is incubated at room temperature.Use confocal microscope（Olympus FV1000）Observe cell dyeing situation.It can be seen that After AAV9-SaCas9-sgRNA5 is treated, mouse movement neuronal cell degenerative condition compares compared with AAV9-SaCas9-sgLacZ Group is obviously improved.
After AAV9-SaCas9-sgRNA5 is treated, mouse movement neuronal cell degenerative condition testing result：
（1）For SOD1G93A transgenic mices after AAV9-SaCas9-sg5 is treated, immuno-fluorescence assay Neu N are positive Motor neuron and IBA1 positive microglia cell morphology degenerative conditions, the degenerative conditions of motor neuron cell obtain It is obviously improved, sees Fig. 4（A）；
（2）Double dye SaCas9 and SOD1 mutant, when confocal microscope observation is last eventually in mouse movement neuron SaCas9 and SOD1 expressions, the SOD1 mutant expressions in SaCas9 positive cells are very low, see Fig. 4（C）.
Described above is only the preferred embodiment of the present invention, it is noted 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.
Bibliography
[1] Qiao Lei, Li Xiaoguang, Liu Mingsheng, the clinical observation China of Cui Li English ALS natural histories Medical journal 2014,94 (9): 674-677.
[2] Bucchia M, Ramirez A, Parente V, Simone C, Nizzardo M, Magri F, et al. Therapeutic Development in Amyotrophic Lateral Sclerosis. ClinTher. 2015; 37(3):668-680.
[3] Cirulli ET, Lasseigne BN, Petrovski S, Sapp PC, Dion PA, Leblond CS,et al. Exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways. Science. 2015; 347(6229):1436-1441.
[4] Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014; 159(2):440-455.
[5] Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR- Cas9 for genome engineering. Cell. 2014;157(6):1262-1278.
[6] Zhang F, Wen Y, Guo X. CRISPR/Cas9 for genome editing: progress, implications and challenges. Hum Mol Genet. 2014;23(R1):R40-R46.
[7] Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2014;343(6166):84-87.
[8] Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-2308.
[9] Lu Y. Recombinant adeno-associated virus as delivery vector for gene therapy-a review. Stem Cells Dev. 2004; 13:133-45.
[10] Daly TM. Overview of adeno-associated viral vectors. Methods Mol Biol. 2004, 246:157-65.
[11] Long C, McAnally JR, Shelton JM, Mireault AA, Bassel-Duby R, Olson EN. Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science. 2014;345(6201):1184-1188.
[12] Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33(1):102-106.
[13] Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520(7546): 186-191.
[14] Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014; 159(2):440-455.
[15] Dirren E, Aebischer J, Rochat C, Towne C, Schneider BL, Aebischer P. SOD1 silencing in motoneurons or glia rescues neuromuscular function in ALS mice.AnnClinTransl Neurol. 2015;2(2):167-184.
[16] Benkhelifa-Ziyyat S, Besse A, Roda M, Duque S, Astord S, Carcenac R,et al. Intramuscular scAAV9-SMN injection mediates widespread gene delivery to the spinal cord and decreases disease severity in SMA mice. Mol Ther. 2013;21(2):282-290.
[17] Duque S, Joussemet B, Riviere C, Marais T, Dubreil L, DouarAM, et al. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons. Mol Ther. 2009;17(7):1187-1196.
[18] Samaranch L, Salegio EA, San SW, Kells AP, Bringas JR, Forsayeth J,et al. Strong cortical and spinal cord transduction after AAV7 and AAV9 delivery into the cerebrospinal fluid of nonhuman primates. Hum Gene Ther. 2013;24(5):526-532.
[19] Maclaren RE, Groppe M, Barnard AR, Cottriall CL, Tolmachova T, Seymour L, et al. Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial.Lancet. 2014, 383(9923): 1129-1137.
[20] Nathwani AC, Tuddenham EG, Rangarajan S, Rosales C, McIntosh J, Linch DC, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med. 2011;365(25):2357-2365.
[21] Kim JY, Grunke SD, Levites Y, Golde TE, Jankowsky JL. Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. J Vis Exp. 2014 Sep 15;(91): 51863. doi: 10.3791/51863.
SEQ ID
No.1
5'-GGCCCACCGTGTTTTCTGGATA-3'
No.2
5'-GACGGCGCTAGGATCATCAACGAAACCCAGCATCTACACAATGTAGCTCAAGTATTCTGGTCACAGAATA CAACGAAACCCAGCATCTACACAATGTAGCTCAAGATGATCCTAGCGCCGTCTT-3'
No.3
5'-CTATATAAGCAGAGCTCTCTGGCTAACTACCGGTGCCACCATGGCCCCAAAGAAGAAGCGGAAGGTCGGT ATCCACGGAGTCCCAGCAGCCAAGCGGAACTACATCCTGGGCCTGGACATCGGCATCACCAGCGTGGGCTACGGCAT CATCGACTACGAGACACGGGACGTGATCGATGCCGGCGTGCGGCTGTTCAAAGAGGCCAACGTGGAAAACAACGAGG GCAGGCGGAGCAAGAGAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGCTGCTG TTCGACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTACGAGGCCAGAGTGAAGGGCCTGAG CCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGCCCTGCTGCACCTGGCCAAGAGAAGAGGCGTGCACAACGTGAACG AGGTGGAAGAGGACACCGGCAACGAGCTGTCCACCAAAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAA TACGTGGCCGAACTGCAGCTGGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAGACCAG CGACTACGTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTCATCGACA CCTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACCTGGCGAGGGCAGCCCCTTCGGCTGGAAGGAC ATCAAAGAATGGTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGGAACTGCGGAGCGTGAAGTACGCCTA CAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAATCTCGTGATCACCAGGGACGAGAACGAGAAGCTGGAAT ATTACGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCAGAAGAAGAAGCCCACCCTGAAGCAGATCGCCAAAGAA ATCCTCGTGAACGAAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCA-3'
No.4
5'-TGGTCTTGCTGATTCTGCCCTTGCCCTTGGCCAGATTCAGGATGTGCTTCTTGAAGGTTTCGTAGCTGAT CTTGCTGTCGCTGCTGCTCAGGTACTGGAATGGGGTCCGGTTGCCCTTCTTGCTGTTTTCTTCCTGCTTCACGAGCA CCTTGTTGTTGAAGCTGTTGTCGAAGGACACGCTTCTGGGGATGATGTGGTCCACCTCATAGTTGAAGGGGTTGTTC AGCAGATCTTCCAGAGGGATGGCTTCCAGGCTGTACAGGCACTTGCCTTCCTGCATGTCGTGCAGCTTGATCTTCTC GATCAGGTACTTGGCGTTCTCTTTGCCGGTGGTCCGGATGATTTCCTCGATCCGCTCGTTGGTCTGCCGGTTCCGCT TCTGCATCTCGTTGATCATTTTCTGGGCGTCCTTGGAGTTCTTCTCGCGGGCCAGCTCGATAATGATGTCGTTGGGC AGGCCGTACTTCTTGATGATGGCGTTGATCACTTTGATGCTCTGGATGAAGCTTCTCTTCACGACGGGGCTCAGGAT GAAGTCGTCCACCAGGGTGGTGGGGATCTCTTTCTGCTGGGACAGGTCCACCTTCTTGGGCACCAGCTTCAGCCGGT TGAAGATAGCGATCTGGTTGTCGTTGGTGTGCCACAGCTCGTCCAGGATCAGGTTGATGGCCTTCAGGCTCAGGTTG TGGGTGCCGGTATAGCCCTTCAGATTAGAGATCTGCTCGATCTCTTCCTGGGTCAGCTCGGAGTTCAGATTGGTCAG TTCTTCCTGGATGTCCTCGCTGCTCTGGTAGATGGTCAGGATCTTGGCAATCTGATCCAGCAGCTCGGCGTTCTCAA TAATCTCTTTCCGGGCGGTAATGTCCTTGATGTCGTGGTACACCTTCAGGTTGGTGAACTCGGGCTTGCCGGTGCTG GTCACTCTGTAGCCCTTAATATCCTCTTCGTTCACGAGGAT-3'
No.5
5'-GAATTCTTAAGCGTAATCTGGAACATCGTATGGGTAAGCGTAATCTGGAACATCGTATGGGTAAGCGTAA TCTGGAACATCGTATGGGTAGGATCCCTTTTTCTTTTTTGCCTGGCCGGCCTTTTTCGTGGCCGCCGGCCTTTTGCC CTTTTTGATGATCTGAGGGTGCTTCTTAGATTTCACTTCATACAGGTTGCCCAGAATGTCTGTGCTGTACTTCTTAA TGCTCTGGGTCTTGGAGGCGATTGTCTTAATGATCCTGGGGGGCCTCTTGTCGTTCATGTTTTCCAGGTACTCGCGG TAGGTGATGTCGATCATGTTCACTTCGATCCGGTTCAGCAGGTCGTTGTTCACGCCGATCACTCTATACAGCTCGCC GTTGATCTTGATCAGATCGTTGTTGTAGAAGGAGGCGATAAACTCGGCCTGGTTGCTGATCTTCTTCAGCTTCTTAG CTTCCTCATAGCACTTGCTATTCACTTCGTAGTAGTTTTCTTTTTTGATCACATCCAGATTCTTCACGGTCACGAAC TTGTACACGCCATTGTCCAGGTACACGTCGAATCTGTAGGGCTTCAGGGACAGCTTCACGACCTTGTTTCTGCTGTT GGGGTAGTCGTCGGTGATGTCCAGATGGGCGTTCAGTTTGTTGCCGTAATACTTAATCTTCTTGATCACGGGGCCGT TGTCCTTTTTGGAGTACTTGGTCAGGTAGTTCCCGGTTTCCTCGTAGTACTTGTACAGGGGATTCTTCTCGTCGCCG TACTGTTCCATAATCAGCTTCAGTTTCTGGTAGGTCTGGGGGTCGTGGTGGTACATCAGCAGCTTTTCGGGGCTCTT GTTGATCAGCTTTTTCAGCTTGTCATTGTCCTTGTCGTACAGGCCGTTCAGATTGTTCACGATCAGGGTGTTGCCCT TGTCGTCCTTCCGGGTGGAGTACAGGGTGTCGTT-3'
No.6
5'-CATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTA CAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGAGACC ACGGCAGGTCTCAGTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAAC TTGTTGGCGAGATTTTTGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCT CACNGAGGCCGGGCGACCAAAGGT-3'

Claims (11)

1. a kind of sgRNA, it is characterised in that it has：
（Ⅰ）, nucleotide sequence as shown in SEQ ID No.1；Or
（Ⅱ）, nucleotide sequence as shown in SEQ ID No.1 complementary series；Or
（Ⅲ）And（Ⅰ）Or（Ⅱ）Nucleotide sequence coded same protein, but because of the degeneracy of genetic code and with（Ⅰ）Or （Ⅱ）The different sequence of nucleotide sequence；Or
（Ⅳ）And（Ⅰ）Or（Ⅱ）Or（Ⅲ）The sequence of the sequence at least 70% homology.
2. sgRNA according to claim 1 is used for the purposes for editing SOD1 genes.
3. a kind of composition, it is characterised in that including AAV9 and SaCas9 and sgRNA as claimed in claim 1.
A kind of 4. recombinant vector expression unit, it is characterised in that including：
（1）Encode the nucleotide sequence of albumen needed for AAV9 duplications；And/or
（2）Encode the nucleotide sequence of SaCas9 albumen；And/or
（3）Encode sgRNA as claimed in claim 1 DNA；
（4）Recombinant vector expression unit structure is ITR-CMV promoter-SaCas9-BGH poly (A) signal-U6 promoter-SOD1 sgRNA-ITR。
5. the construction method of recombinant vector expression unit according to claim 4, it is characterised in that replicate coding AAV9 The DNA of the nucleotide sequence of required albumen and the nucleotide sequence and coding RNA as claimed in claim 1 of coding SaCas9 albumen It is connected in carrier, construction of expression vector.
6. construction method according to claim 5, it is characterised in that the carrier is plasmid or virus.
7. construction method according to claim 6, it is characterised in that the viral including but not limited to adeno-associated virus, Slow virus, adenovirus, herpes simplex virus, rabies viruses and related derivatives.
8. a kind of gene therapy mode, it is characterised in that including described in sgRNA as claimed in claim 1 and claim 3 Composition.
9. gene therapy mode according to claim 8, it is characterised in that the gene therapy mode is intracerebroventricular weight In the SOD1 genes of body editor mutation after group virus.
10. described in the recombinant expression carrier, claim 8 or 9 described in composition according to claim 3, claim 5 Gene therapy mode prepare treat amyotrophic lateral sclerosis in application.
11. application according to claim 10, it is characterised in that the amyotrophic lateral sclerosis is moved back for nerve Row disease.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110195057A (en) * 2018-02-26 2019-09-03 百奥赛图江苏基因生物技术有限公司 The preparation method and application of the genetically modified non-human animal of Hr gene or its filial generation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104769112A (en) * 2012-11-01 2015-07-08 菲克特生物科学股份有限公司 Methods and products for expressing proteins in cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104769112A (en) * 2012-11-01 2015-07-08 菲克特生物科学股份有限公司 Methods and products for expressing proteins in cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FU Y: "Improving CRISPR-Cas nuclease specificity using truncated guide RNAs", 《NAT BIOTECHNOL.》 *
谢胜松: "CRISPR/Cas9系统中sgRNA 设计与脱靶效应评估", 《遗传》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110195057A (en) * 2018-02-26 2019-09-03 百奥赛图江苏基因生物技术有限公司 The preparation method and application of the genetically modified non-human animal of Hr gene or its filial generation

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