CN111304245B - Method for injecting virus into specific brain region of animal for gene editing based on non-therapeutic purpose of MECP2 gene - Google Patents

Method for injecting virus into specific brain region of animal for gene editing based on non-therapeutic purpose of MECP2 gene Download PDF

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CN111304245B
CN111304245B CN201911260388.8A CN201911260388A CN111304245B CN 111304245 B CN111304245 B CN 111304245B CN 201911260388 A CN201911260388 A CN 201911260388A CN 111304245 B CN111304245 B CN 111304245B
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virus
brain region
gene
specific brain
injected
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CN111304245A (en
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吴诗昊
李霄
程田林
秦冬冬
吴晶
胡新天
仇子龙
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Kunming Institute of Zoology of CAS
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Abstract

The invention discloses a method for injecting viruses into specific brain regions of animals for gene editing based on the non-therapeutic purpose of MECP2 genes, which utilizes a brain stereotactic injection pump to directly inject the viruses into the specific brain regions of non-human primates; the injection quantity is 1m 3 The virus was injected in an amount of 1uL,the titer of injected virus was 10 x 10 13 vg/mL. According to the method, viruses are directly injected into specific brain regions of non-human primates for gene editing, a transgenic monkey model with the disease phenotype can be obtained in a short time, and the urgent needs of research on Autism Spectrum Disorder (ASD) diseases in the current stage are solved.

Description

Method for injecting virus into specific brain region of animal for gene editing based on non-therapeutic purpose of MECP2 gene
Technical Field
The invention belongs to the field of transgenic technology and modeling of non-human primate animals, and particularly relates to a method for constructing an ASD disease model of the non-human primate animals by injecting viruses into specific brain regions of the non-human primate animals for gene editing based on the non-therapeutic purpose of MECP2 genes.
Background
Autism spectrum disorder (Autism Spectrum Disorder, ASD) is characterized by patient suffering from autism-like behavior, loss of motor control, irregular breathing, and skeletal problems. Children suffering from ASD-like diseases cannot participate in normal educational processes, thereby having a great influence on their normal social integration and social functioning after adulthood. At present, no effective treatment method for ASD diseases exists, so that ASD has serious negative effects on family happiness and social harmony development, and is widely paid attention to all communities.
In recent years, the prevalence of ADS has been increasing and has become the first disease that is detrimental to mental health in teenagers. The prevalence of domestic ASD remains to be published, but even if the prevalence is less than one percent, the ASD patients in china will go over millions. The pathogenesis of ASD is not completely understood at present, but there is sufficient evidence that genetic factors are important risk factors for this disease: the ASD incidence risk of the newborn is 10 times higher than that of other common families in the existing ASD infant families, and the co-morbidity of twins in the same egg is as high as 70% -90%. In addition, patients with various monogenic genetic diseases also exhibit core symptoms of ASD, suggesting that genetic mutations may lead to the appearance of core symptoms of ASD. Since ASD is a highly inherited disease, the use of susceptibility genes to construct disease models is particularly important in exploring the pathogenesis of the disease.
At present, only a few embryo ASD macaque models are aimed at macaques, but (1) the individual genetic background of the macaques is large, and the problem of chimerism of embryo transgenic technology cannot be solved, so that the macaques based on embryo transgenic technology are difficult to help us observe and research the etiology and pathogenesis of ASD at present. (2) The natural maturation time of the macaque is 5-6 years, and the gestation period needs 6 months, which belongs to single-fetal reproductive animals, so that the current stage of gene operation of the macaque which utilizes the mating of transgenic macaques to obtain homozygotes is difficult to realize. (3) Transgenic monkey based on embryo transgenic technology has high mortality, low success rate, poor stability of transgene retention and extremely long onset time, and a stable transgenic monkey model is difficult to establish at present. Even if the model of the monkey is successfully established, the model is difficult to reproduce in large quantities and the maintenance cost is extremely high so that the urgent research requirements at the present stage are difficult to meet.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for injecting viruses into specific brain areas of non-human primates for gene editing based on the non-therapeutic purpose of MECP2 genes.
The invention adopts the technical scheme that: a method for injecting virus into specific brain region of non-human primate for gene editing based on non-therapeutic purpose of MECP2 gene, directly injecting virus into specific brain region of non-human primate with microinjector; the injection quantity is 1m 3 1uL of injected virus with a titer of 10×10 13 vg/mL。
Further, the non-human primate is a macaque or cynomolgus monkey; the specific brain region of the non-human primate is the hippocampal brain region. The sgRNA capable of expressing the targeted macaque MECP2 gene is shown in a sequence table.
Further, the virus is injected at a speed of 600-800 nL/min, and the needle is left for 10-15 min after injection. The virus is adeno-associated virus (AAV).
Further, the microinjector is placed on an injection pump of a stereotactic instrument and injected by a multi-point array injection method under the guidance of MRI positioning.
The invention has the following remarkable characteristics: the problems of genetic difference among macaque individuals and chimera of embryo transgenic technology are effectively avoided, and the defects of high mortality, low success rate, poor stability of transgene retention, long molding time and the like of the traditional embryo transgenic technology are overcome. The exogenous gene is directly injected into a specific brain region of a non-human primate, so that a transgenic monkey model can be obtained in a short time, and the urgent ASD (autism spectrum disorder) disease research requirement at the present stage is solved.
Drawings
Fig. 1 is a schematic diagram of the injection principle of virus into specific brain regions of non-human primate for non-therapeutic purposes based on MECP2 gene according to the embodiment of the invention.
FIG. 2 is a schematic diagram of MECP2 gene editing method in the embodiment of the present invention.
Detailed Description
At present, the injection of animal models aiming at the diseases is concentrated on rodents mainly comprising mice, the relativity of the rodents and human beings is far, the correspondingly developed cerebral cortex is lacking, the social behavior is simple, and the cerebral development process is far different from that of the human beings. (Johnson, wang et al 2015), but non-human primates, particularly macaques, have a genetic similarity of up to 93.5% with humans, and are very similar to humans in physiological structure, development process, brain function and performance. More importantly, the macaque is a natural social animal, and has obvious advantages compared with other animals in researching diseases such as ASD and the like with typical social barriers.
Therefore, the present invention provides a method for directly injecting virus into specific brain regions of non-human primate for gene editing, and the following description will be given by taking macaque as an example.
As shown in fig. 1 and 2, in this embodiment, adeno-associated virus (AAV) is directly injected into a specific brain region of macaque, and the MECP2 gene of the macaque injected into the brain region is knocked out by using CRISPR/Cas9 gene editing technology, so as to construct a transgenic macaque capable of simulating ASD core symptoms. The specific operation steps are as follows:
(1) Before virus injection, social behavior acquisition is carried out on the macaque by using an active body movement recorder and a video analysis method in a monkey group living on the macaque, and the method comprises the following steps: autonomous activities, reactive exercises, environmental exploration, social interaction, clashing behavior, conflicting behavior, sleep rhythms, and the like. Social behavior baseline of macaque virus in monkey group before injection is obtained, and single cage brain cognitive function test is combined, such as: inward Testing (HIT), brinkman board test, social re-ward, gaze communication, etc. are further used for collecting various behavioral indexes and brain functions of the macaque.
(2) The specific brain region of the injection, in this example the hippocampal brain region, is located using an MRI (magnetic resonance imaging) deep brain structure accurate locating technique. Under the guidance of MRI positioning, the virus is precisely injected into a specific brain region of macaque by using a multi-point array injection method.
Specifically, the method for the transgene described in this example: firstly, anaesthetizing macaque, opening scalp and scalp surface tissue layer by layer, and positioning at the positionDrilling skull; then the virus is extracted by a micro-injector and is placed on an injection pump of a stereotactic instrument, and the injection is carried out at the speed of 600-800 nL/min (preferably the injection speed of 800 nL/min), the needle is left for 10-15 min (minutes) after the injection, and the injection quantity is 1m each time 3 1uL of injected virus with a titer of 10×10 13 vg/mL. The viral vector for the transgene is AAV (adeno-associated virus).
(3) After virus injection, the macaque is subjected to behavior collection and analysis again, and compared with the previously collected basic behaviors and the macaque of an empty virus injection control group, the macaque is observed to show which behavior changes appear before and after transgene, and whether symptoms of ASD diseases appear or not. After acquisition of behavioural data, part of macaque was sacrificed for detection of hippocampal and peripheral brain region histology and gene mutation, and changes in macaque synapses were focused using histology and electron microscopy.
In addition, in embodiments of the invention, the non-human primate may also be selected to be a cynomolgus monkey, the method is a CRISPR/CAS9 technology knockout of MECP2 gene, and the animal model is a transgenic non-human primate model of human autism spectrum disorder.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various changes and modifications can be made without departing from the principles of the invention, and such changes and modifications are intended to be included within the scope of the invention.
<110> Kunming animal institute of China academy of sciences
<120> method for gene editing by injecting virus into specific brain region of animal based on non-therapeutic purpose of MECP2 gene
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 26
<212> DNA
<213> artificial sequence
<223> DNA sequence of sgRNA sgRNA-MECP2-a capable of expressing targeted macaque MECP2 gene with base for vector construction
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caccgaccac caccatcacc accact 26
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<212> DNA
<213> artificial sequence
<223> expression of the complementary DNA sequence of sgRNA sgRNA-MECP2-a targeting the macaque MECP2 gene with bases for vector construction
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aaacagtggt ggtgatggtg gtggtc 26
<210> 3
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<213> artificial sequence
<223> DNA sequence of sgRNA sgRNA-MECP2-b capable of expressing targeted macaque MECP2 gene with base for vector construction
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caccgcctgg ggctcagggg ggctgg 26
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<223> expression of the complementary DNA sequence of sgRNA-MECP2-b targeting the macaque MECP2 gene with bases for vector construction
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aaacccagcc cccctgagcc ccaggc 26
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<223> vector p-sgRNA-MECP2-a sequence capable of expressing sgRNA sgRNA-MECP2-a targeting macaque MECP2 gene
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acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 60
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 120
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 180
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 240
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 300
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 360
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 420
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 480
ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 540
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 600
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 660
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 720
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 780
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 840
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 900
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 960
ggcttccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1020
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1080
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1140
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1200
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1260
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1320
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1380
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1440
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1500
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1560
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1620
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1680
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1740
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1800
aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 1860
gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 1920
tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 1980
gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 2040
agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg 2100
cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 2160
cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga 2220
gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 2280
atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag 2340
cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 2400
acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 2460
tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 2520
cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa 2580
ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg 2640
ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 2700
cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcgagg gcctatttcc 2760
catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa 2820
tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct 2880
tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac 2940
ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgacca 3000
ccaccatcac caccactgtt ttagtactct ggaaacagaa tctactaaaa caaggcaaaa 3060
tgccgtgttt atctcgtcaa cttgttggcg agatttttgg tacctcccca gcatgcctgc 3120
tattctcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat 3180
gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag 3240
tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac 3300
tagaaggcac agtcgaggct gatcagcgag ctctaggaat tcttactttc ccatcaacct 3360
gggtccaagc tcgttggcga agccgctagc gtaatctgga acatcgtatg ggtaggatcc 3420
ctttttcttt tttgcctggc cggccttttt cgtggccgcc ggccttttgc cctttttgat 3480
gatctgaggg tgcttcttag atttcacttc atacaggttg cccagaatgt ctgtgctgta 3540
cttcttaatg ctctgggtct tggaggcgat tgtcttaatg atcctggggg gcctcttgtc 3600
gttcatgttt tccaggtact cgcggtaggt gatgtcgatc atgttcactt cgatccggtt 3660
cagcaggtcg ttgttcacgc cgatcactct atacagctcg ccgttgatct tgatcagatc 3720
gttgttgtag aaggaggcga taaactcggc ctggttgctg atcttcttca gcttcttagc 3780
ttcctcatag cacttgctat tcacttcgta gtagttttct tttttgatca catccagatt 3840
cttcacggtc acgaacttgt acacgccatt gtccaggtac acgtcgaatc tgtagggctt 3900
cagggacagc ttcacgacct tgtttctgct gttggggtag tcgtcggtga tgtccagatg 3960
ggcgttcagt ttgttgccgt aatacttaat cttcttgatc acggggccgt tgtccttttt 4020
ggagtacttg gtcaggtagt tcccggtttc ctcgtagtac ttgtacaggg gattcttctc 4080
gtcgccgtac tgttccataa tcagcttcag tttctggtag gtctgggggt cgtggtggta 4140
catcagcagc ttttcggggc tcttgttgat cagctttttc agcttgtcat tgtccttgtc 4200
gtacaggccg ttcagattgt tcacgatcag ggtgttgccc ttgtcgtcct tccgggtgga 4260
gtacagggtg tcgttaatca gctctctatt aggcttcttg tccacccggt ggctgtactt 4320
gtagtccttg aagtccttaa tgtgcttgat ctggtggggg gtgatgaaga tctctttgta 4380
ctcctgctcg gtttcgatct cgggcatgct ctcggcctgc ttttcctcga acatctggtt 4440
ttccatcact tttttggcct tgtccagttt cttccactct ttgaagatga aatcggcgtt 4500
ggcaatgatc agggcgtcct cggcgtggtg cttgtacccc ttgttccgct ctttcttaaa 4560
cttccacttc cgccgcagaa agctggtgaa gccgccattg atggacttca ctttcacgtc 4620
caggttgttc actctgaagt agctccgcag caggttcatc aggcctctgg tggcgtatct 4680
ggtatccacc aggttccggt tgatgaagtc tttctgcacg gagaacctgt tgatgtcccg 4740
ttcttccagc agatactctt tcttggtctt gctgattctg cccttgccct tggccagatt 4800
caggatgtgc ttcttgaagg tttcgtagct gatcttgctg tcgctgctgc tcaggtactg 4860
gaatggggtc cggttgccct tcttgctgtt ttcttcctgc ttcacgagca ccttgttgtt 4920
gaagctgttg tcgaaggaca cgcttctggg gatgatgtgg tccacctcat agttgaaggg 4980
gttgttcagc agatcttcca gagggatggc ttccaggctg tacaggcact tgccttcctg 5040
catgtcgtgc agcttgatct tctcgatcag gtacttggcg ttctctttgc cggtggtccg 5100
gatgatttcc tcgatccgct cgttggtctg ccggttccgc ttctgcatct cgttgatcat 5160
tttctgggcg tccttggagt tcttctcgcg ggccagctcg ataatgatgt cgttgggcag 5220
gccgtacttc ttgatgatgg cgttgatcac tttgatgctc tggatgaagc ttctcttcac 5280
gacggggctc aggatgaagt cgtccaccag ggtggtgggg atctctttct gctgggacag 5340
gtccaccttc ttgggcacca gcttcagccg gttgaagata gcgatctggt tgtcgttggt 5400
gtgccacagc tcgtccagga tcaggttgat ggccttcagg ctcaggttgt gggtgccggt 5460
atagcccttc agattagaga tctgctcgat ctcttcctgg gtcagctcgg agttcagatt 5520
ggtcagttct tcctggatgt cctcgctgct ctggtagatg gtcaggatct tggcaatctg 5580
atccagcagc tcggcgttct caataatctc tttccgggcg gtaatgtcct tgatgtcgtg 5640
gtacaccttc aggttggtga actcgggctt gccggtgctg gtcactctgt agcccttaat 5700
atcctcttcg ttcacgagga tttctttggc gatctgcttc agggtgggct tcttcttctg 5760
cttgaacacg ttctcgatga tctggaactt ctcgtaatat tccagcttct cgttctcgtc 5820
cctggtgatc acgagattgt tcaggtcgtt cagggcgttg tacaggtcgg cgttgtaggc 5880
gtacttcacg ctccgcagtt cctcggggaa gtaggtgcag tggcccatca gcatctcgta 5940
ccattctttg atgtccttcc agccgaaggg gctgccctcg ccaggtccct catagtaggt 6000
ccgccgggtt tccagcaggt cgatgtaggt gtcgatgaag ctctggtcca gctggtggta 6060
ggccttctgc accttcagca gctgtttggc ttctttcacg tagtcgctgg tcttgaatct 6120
gttgatgctg ccccgcactt cgccgtcttt cttcagccgt tccagctgca gttcggccac 6180
gtatttctct tccagggcct tgctgttccg gctgatctgc tctttggtgg acagctcgtt 6240
gccggtgtcc tcttccacct cgttcacgtt gtgcacgcct cttctcttgg ccaggtgcag 6300
cagggcggca gagaactctt cctcgctcag cttctggctc aggcccttca ctctggcctc 6360
gtaggggttg atgccgctca gctcgctgtg gtcggtcagc aggttgtagt cgaacagcag 6420
cttcttcact ctctggattc tatgccgcct ccgccgcttc agccttctgg cgcctctctt 6480
gctccgcctg ccctcgttgt tttccacgtt ggcctctttg aacagccgca cgccggcatc 6540
gatcacgtcc cgtgtctcgt agtcgatgat gccgtagccc acgctggtga tgccgatgtc 6600
caggcccagg atgtagttcc gcttggctgc tgggactccg tggataccga ccttccgctt 6660
cttctttggg gccatggtgg caccggtacc cggggatccg gtaccttctc gactgcgctc 6720
tcaggcacga cacgactcct ccgctgccca ccgcagactg aggcagcgct gagtcgccgg 6780
cgccgcagcg cagatggtcg cgcccgtgcc cccctatctc gcgcctcgcg tggtgcggtc 6840
cggctgggcc ggcggcggcg cggacgcgac caaggtggcc gggaagggga gtttgcgggg 6900
gaccggcgag tgacgtcagc gcgccttcag tgctgaggcg gcggtggcgc gcgccgccag 6960
gcgggggcga aggcactgtc cgcggtgctg aagctggcag tgcgcacgcg cctcgccgca 7020
tcctgtttcc cctccccctc tctgataggg gatgcgcaat ttggggaatg ggggttgggt 7080
gcttgtccag tgggtcgggg tcggtcgtca ggtaggcacc cccaccccgc ctcatcctgg 7140
tcctaaaacc cacttgcact catacgcagg gccctctgca gtctagaggc cgcaggaacc 7200
cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 7260
accaaaggtc gcccgacgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc 7320
agg 7323
<210> 6
<211> 7323
<212> DNA
<213> artificial sequence
<223> vector p-sgRNA-MECP2-b sequence capable of expressing sgRNA sgRNA-MECP2-b targeting macaque MECP2 gene
<400> 6
acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt 60
ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt 120
ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc 180
gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa 240
gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct 300
ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta 360
actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg 420
gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc 480
ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta 540
ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 600
gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 660
tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg 720
tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta 780
aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg 840
aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg 900
tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc 960
ggcttccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg 1020
agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg 1080
aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag 1140
gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat 1200
caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc 1260
cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc 1320
ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa 1380
ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac 1440
gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt 1500
cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc 1560
gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa 1620
caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca 1680
tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat 1740
acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa 1800
aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc 1860
gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca 1920
tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc 1980
gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag 2040
agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg 2100
cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 2160
cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga 2220
gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 2280
atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag 2340
cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 2400
acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 2460
tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 2520
cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa 2580
ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg 2640
ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 2700
cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcgagg gcctatttcc 2760
catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttggaattaa 2820
tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct 2880
tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac 2940
ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgcctg 3000
gggctcaggg gggctgggtt ttagtactct ggaaacagaa tctactaaaa caaggcaaaa 3060
tgccgtgttt atctcgtcaa cttgttggcg agatttttgg tacctcccca gcatgcctgc 3120
tattctcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat 3180
gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag 3240
tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac 3300
tagaaggcac agtcgaggct gatcagcgag ctctaggaat tcttactttc ccatcaacct 3360
gggtccaagc tcgttggcga agccgctagc gtaatctgga acatcgtatg ggtaggatcc 3420
ctttttcttt tttgcctggc cggccttttt cgtggccgcc ggccttttgc cctttttgat 3480
gatctgaggg tgcttcttag atttcacttc atacaggttg cccagaatgt ctgtgctgta 3540
cttcttaatg ctctgggtct tggaggcgat tgtcttaatg atcctggggg gcctcttgtc 3600
gttcatgttt tccaggtact cgcggtaggt gatgtcgatc atgttcactt cgatccggtt 3660
cagcaggtcg ttgttcacgc cgatcactct atacagctcg ccgttgatct tgatcagatc 3720
gttgttgtag aaggaggcga taaactcggc ctggttgctg atcttcttca gcttcttagc 3780
ttcctcatag cacttgctat tcacttcgta gtagttttct tttttgatca catccagatt 3840
cttcacggtc acgaacttgt acacgccatt gtccaggtac acgtcgaatc tgtagggctt 3900
cagggacagc ttcacgacct tgtttctgct gttggggtag tcgtcggtga tgtccagatg 3960
ggcgttcagt ttgttgccgt aatacttaat cttcttgatc acggggccgt tgtccttttt 4020
ggagtacttg gtcaggtagt tcccggtttc ctcgtagtac ttgtacaggg gattcttctc 4080
gtcgccgtac tgttccataa tcagcttcag tttctggtag gtctgggggt cgtggtggta 4140
catcagcagc ttttcggggc tcttgttgat cagctttttc agcttgtcat tgtccttgtc 4200
gtacaggccg ttcagattgt tcacgatcag ggtgttgccc ttgtcgtcct tccgggtgga 4260
gtacagggtg tcgttaatca gctctctatt aggcttcttg tccacccggt ggctgtactt 4320
gtagtccttg aagtccttaa tgtgcttgat ctggtggggg gtgatgaaga tctctttgta 4380
ctcctgctcg gtttcgatct cgggcatgct ctcggcctgc ttttcctcga acatctggtt 4440
ttccatcact tttttggcct tgtccagttt cttccactct ttgaagatga aatcggcgtt 4500
ggcaatgatc agggcgtcct cggcgtggtg cttgtacccc ttgttccgct ctttcttaaa 4560
cttccacttc cgccgcagaa agctggtgaa gccgccattg atggacttca ctttcacgtc 4620
caggttgttc actctgaagt agctccgcag caggttcatc aggcctctgg tggcgtatct 4680
ggtatccacc aggttccggt tgatgaagtc tttctgcacg gagaacctgt tgatgtcccg 4740
ttcttccagc agatactctt tcttggtctt gctgattctg cccttgccct tggccagatt 4800
caggatgtgc ttcttgaagg tttcgtagct gatcttgctg tcgctgctgc tcaggtactg 4860
gaatggggtc cggttgccct tcttgctgtt ttcttcctgc ttcacgagca ccttgttgtt 4920
gaagctgttg tcgaaggaca cgcttctggg gatgatgtgg tccacctcat agttgaaggg 4980
gttgttcagc agatcttcca gagggatggc ttccaggctg tacaggcact tgccttcctg 5040
catgtcgtgc agcttgatct tctcgatcag gtacttggcg ttctctttgc cggtggtccg 5100
gatgatttcc tcgatccgct cgttggtctg ccggttccgc ttctgcatct cgttgatcat 5160
tttctgggcg tccttggagt tcttctcgcg ggccagctcg ataatgatgt cgttgggcag 5220
gccgtacttc ttgatgatgg cgttgatcac tttgatgctc tggatgaagc ttctcttcac 5280
gacggggctc aggatgaagt cgtccaccag ggtggtgggg atctctttct gctgggacag 5340
gtccaccttc ttgggcacca gcttcagccg gttgaagata gcgatctggt tgtcgttggt 5400
gtgccacagc tcgtccagga tcaggttgat ggccttcagg ctcaggttgt gggtgccggt 5460
atagcccttc agattagaga tctgctcgat ctcttcctgg gtcagctcgg agttcagatt 5520
ggtcagttct tcctggatgt cctcgctgct ctggtagatg gtcaggatct tggcaatctg 5580
atccagcagc tcggcgttct caataatctc tttccgggcg gtaatgtcct tgatgtcgtg 5640
gtacaccttc aggttggtga actcgggctt gccggtgctg gtcactctgt agcccttaat 5700
atcctcttcg ttcacgagga tttctttggc gatctgcttc agggtgggct tcttcttctg 5760
cttgaacacg ttctcgatga tctggaactt ctcgtaatat tccagcttct cgttctcgtc 5820
cctggtgatc acgagattgt tcaggtcgtt cagggcgttg tacaggtcgg cgttgtaggc 5880
gtacttcacg ctccgcagtt cctcggggaa gtaggtgcag tggcccatca gcatctcgta 5940
ccattctttg atgtccttcc agccgaaggg gctgccctcg ccaggtccct catagtaggt 6000
ccgccgggtt tccagcaggt cgatgtaggt gtcgatgaag ctctggtcca gctggtggta 6060
ggccttctgc accttcagca gctgtttggc ttctttcacg tagtcgctgg tcttgaatct 6120
gttgatgctg ccccgcactt cgccgtcttt cttcagccgt tccagctgca gttcggccac 6180
gtatttctct tccagggcct tgctgttccg gctgatctgc tctttggtgg acagctcgtt 6240
gccggtgtcc tcttccacct cgttcacgtt gtgcacgcct cttctcttgg ccaggtgcag 6300
cagggcggca gagaactctt cctcgctcag cttctggctc aggcccttca ctctggcctc 6360
gtaggggttg atgccgctca gctcgctgtg gtcggtcagc aggttgtagt cgaacagcag 6420
cttcttcact ctctggattc tatgccgcct ccgccgcttc agccttctgg cgcctctctt 6480
gctccgcctg ccctcgttgt tttccacgtt ggcctctttg aacagccgca cgccggcatc 6540
gatcacgtcc cgtgtctcgt agtcgatgat gccgtagccc acgctggtga tgccgatgtc 6600
caggcccagg atgtagttcc gcttggctgc tgggactccg tggataccga ccttccgctt 6660
cttctttggg gccatggtgg caccggtacc cggggatccg gtaccttctc gactgcgctc 6720
tcaggcacga cacgactcct ccgctgccca ccgcagactg aggcagcgct gagtcgccgg 6780
cgccgcagcg cagatggtcg cgcccgtgcc cccctatctc gcgcctcgcg tggtgcggtc 6840
cggctgggcc ggcggcggcg cggacgcgac caaggtggcc gggaagggga gtttgcgggg 6900
gaccggcgag tgacgtcagc gcgccttcag tgctgaggcg gcggtggcgc gcgccgccag 6960
gcgggggcga aggcactgtc cgcggtgctg aagctggcag tgcgcacgcg cctcgccgca 7020
tcctgtttcc cctccccctc tctgataggg gatgcgcaat ttggggaatg ggggttgggt 7080
gcttgtccag tgggtcgggg tcggtcgtca ggtaggcacc cccaccccgc ctcatcctgg 7140
tcctaaaacc cacttgcact catacgcagg gccctctgca gtctagaggc cgcaggaacc 7200
cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 7260
accaaaggtc gcccgacgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc 7320
agg 7323

Claims (3)

1. A method for gene editing by injecting a virus into a specific brain region of a non-human primate for non-therapeutic purposes based on the MECP2 gene, characterized by: directly injecting adeno-associated virus into a specific brain region of the macaque, knocking out MECP2 genes of the macaque injected into the brain region by using a CRISPR/Cas9 gene editing technology, and constructing a transgenic macaque capable of simulating ASD core symptoms; under the positioning guidance of MRI, directly injecting the adeno-associated virus into a specific brain region of a non-human primate by using a microinjector by using a multi-point array injection method; the virus is injected at the speed of 600-800 nL/min, the needle is left for 10-15 min after the injection, the injection quantity is 1uL of the injected virus per 1m, and the titer of the injected virus isThe method comprises the steps of carrying out a first treatment on the surface of the The sgRNA capable of expressing the targeted MECP2 gene is shown as SEQ ID NO. 1 and SEQ ID NO. 3, and
wherein the specific brain region of the non-human primate is the hippocampal brain region.
2. The method for gene editing by injecting a virus into a specific brain region of a non-human primate for non-therapeutic purposes based on MECP2 gene of claim 1, wherein the virus is injected at a rate of 800nL/min and the needle is left for 10-15 min after the injection.
3. The method for gene editing by injecting a virus into a specific brain region of a non-human primate for non-therapeutic purposes based on the MECP2 gene of claim 1 wherein the microinjector is placed on a syringe pump of a stereotactic apparatus and injected under the guidance of MRI positioning using a multi-point array injection method.
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