CN105821116A - 一种绵羊mstn基因定向敲除及其影响成肌分化的检测方法 - Google Patents
一种绵羊mstn基因定向敲除及其影响成肌分化的检测方法 Download PDFInfo
- Publication number
- CN105821116A CN105821116A CN201610237236.6A CN201610237236A CN105821116A CN 105821116 A CN105821116 A CN 105821116A CN 201610237236 A CN201610237236 A CN 201610237236A CN 105821116 A CN105821116 A CN 105821116A
- Authority
- CN
- China
- Prior art keywords
- gene
- cas9
- crispr
- knockout
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000000694 effects Effects 0.000 title claims abstract description 80
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 101100448232 Ovis aries MSTN gene Proteins 0.000 title claims abstract description 11
- 230000004070 myogenic differentiation Effects 0.000 title claims description 17
- 108091033409 CRISPR Proteins 0.000 claims abstract description 87
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 27
- 238000003209 gene knockout Methods 0.000 claims abstract description 23
- 238000013461 design Methods 0.000 claims abstract description 12
- 210000000419 skeletal muscle satellite cell Anatomy 0.000 claims abstract description 11
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 4
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 4
- 108020005004 Guide RNA Proteins 0.000 claims description 30
- 108090000623 proteins and genes Proteins 0.000 claims description 30
- 108020004414 DNA Proteins 0.000 claims description 23
- 210000004027 cell Anatomy 0.000 claims description 22
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims description 22
- 241001494479 Pecora Species 0.000 claims description 21
- 238000000338 in vitro Methods 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 14
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 claims description 12
- 230000035772 mutation Effects 0.000 claims description 12
- 102000004190 Enzymes Human genes 0.000 claims description 11
- 108090000790 Enzymes Proteins 0.000 claims description 11
- 108060001084 Luciferase Proteins 0.000 claims description 11
- 239000005089 Luciferase Substances 0.000 claims description 11
- 229950010131 puromycin Drugs 0.000 claims description 11
- 210000002950 fibroblast Anatomy 0.000 claims description 9
- 238000001890 transfection Methods 0.000 claims description 9
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims description 8
- 230000014509 gene expression Effects 0.000 claims description 7
- 239000002502 liposome Substances 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 6
- 230000006698 induction Effects 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- 108700008625 Reporter Genes Proteins 0.000 claims description 5
- 235000003642 hunger Nutrition 0.000 claims description 5
- 230000037351 starvation Effects 0.000 claims description 5
- 210000001057 smooth muscle myoblast Anatomy 0.000 claims description 4
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 108010087236 cobra venom endonuclease Proteins 0.000 claims 2
- 238000013467 fragmentation Methods 0.000 claims 2
- 238000006062 fragmentation reaction Methods 0.000 claims 2
- 230000000869 mutational effect Effects 0.000 claims 2
- 230000029087 digestion Effects 0.000 claims 1
- 239000003814 drug Substances 0.000 claims 1
- 239000001963 growth medium Substances 0.000 claims 1
- 238000003780 insertion Methods 0.000 claims 1
- 230000037431 insertion Effects 0.000 claims 1
- 230000003387 muscular Effects 0.000 claims 1
- 238000004513 sizing Methods 0.000 claims 1
- 238000010186 staining Methods 0.000 claims 1
- 210000001519 tissue Anatomy 0.000 claims 1
- 238000013518 transcription Methods 0.000 claims 1
- 230000035897 transcription Effects 0.000 claims 1
- 239000013598 vector Substances 0.000 abstract description 31
- 238000005516 engineering process Methods 0.000 abstract description 17
- 238000010276 construction Methods 0.000 abstract description 8
- 230000009261 transgenic effect Effects 0.000 abstract description 5
- 241001465754 Metazoa Species 0.000 abstract description 4
- 230000008901 benefit Effects 0.000 abstract description 3
- 238000012215 gene cloning Methods 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 230000024245 cell differentiation Effects 0.000 abstract 1
- 238000010363 gene targeting Methods 0.000 abstract 1
- 238000001976 enzyme digestion Methods 0.000 description 10
- 101150048453 MSTN gene Proteins 0.000 description 9
- 238000003776 cleavage reaction Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 238000010008 shearing Methods 0.000 description 8
- 102000004533 Endonucleases Human genes 0.000 description 7
- 108010042407 Endonucleases Proteins 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 230000007017 scission Effects 0.000 description 7
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 6
- 238000010459 TALEN Methods 0.000 description 6
- 230000001404 mediated effect Effects 0.000 description 6
- 238000012795 verification Methods 0.000 description 6
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 210000004748 cultured cell Anatomy 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 230000004069 differentiation Effects 0.000 description 4
- 238000007877 drug screening Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 210000003205 muscle Anatomy 0.000 description 3
- 210000003098 myoblast Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 102100036912 Desmin Human genes 0.000 description 2
- 108010044052 Desmin Proteins 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 108700005075 Regulator Genes Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000005014 ectopic expression Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000009545 invasion Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 241000252212 Danio rerio Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108010056852 Myostatin Proteins 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种应用CRISPR/Cas9技术对绵羊MSTN基因进行定向敲除，并验证其对骨骼肌卫星细胞分化影响效果的方法，具体内容如下：(1)目的基因克隆；(2)gRNA设计及合成；(3)CRISPR/Cas9基因敲除载体构建；(4)CRISPR/Cas9基因敲除载体外源活性检测；(5)CRISPR/Cas9基因敲除载体内源活性检测；(6)CRISPR/Cas9基因敲除载体敲除效果检测。本发明的优越性在于：1、实验周期较短速；2、方法简单易行，可重复性强，在普通实验室即可进行；3、运用该方法构建的活性载体，毒副作用小，可用于转基因动物的制备与生产；4、该方法非特异剪切较少，显著提高了基因靶向敲除效率高。5、该方法适用性高。
Description
技术领域：
本发明涉及一种绵羊MSTN基因定向敲除及其影响成肌分化的检测方法，属于分子生物学、基因工程学和转基因技术等领域。
技术背景：
目前研究负反馈调节基因的功能主要是运用人工核酸内切酶(Engineeredendonuclease,EEN)技术对基因进行靶向敲除并进行功能性验证，目前应用比较广泛的ENN主要有ZFN(Zinc-fingernuclease,ZFN)和TALEN(Transcriptionactivator-likeeffectornuclease,TALEN)，然而ZFN技术因具有较低特异性的同源二聚体形式而剪切特异性较差、细胞毒性大。TALEN作为第二代EEN技术在特异性剪切方面有了一定的提高，但仍具有较大细胞毒性。这两种技术的实验过程繁琐，设计也较复杂，新型CRISPR/Cas9系统作为第三代ENN技术是模仿细菌用来抵御病毒侵袭/躲避哺乳动物免疫反应的基因系统而诞生的，作为第三代ENN技术的Cas9系统则是这两年兴起的一种快速靶向基因敲除技术，其具有实验过程简单、耗时短和工作量小等优势，且更容易得到纯合子突变体，而且可以在不同的位点同时引入多个突变，具有良好的发展前景，因此，Cas9系统正逐渐地取代ZFN和TALEN技术。目前研究人员已经利用Cas9系统在小鼠、斑马鱼和果蝇等动物中成功的构建了基因敲除模型，并在HEK293和iPS等细胞中实现了基因的敲除并产生了稳定的细胞系，实现了基因的定点修饰及其功能研究。
发明内容：
针对传统ENN技术实验繁琐、效率低、毒性大的缺点，提供一种绵羊MSTN基因定向敲除及其影响成肌分化的检测方法。
本发明包括以下步骤：
(1)目的基因克隆
查询NCBI数据库，获取绵羊目的基因的CDS序列(序列号：NM_001009428)，设计特异性引物，以湖羊肌肉组织总RNA反转录形成的cDNA为模板克隆并获得目的基因全部CDS序列，纯化回收后送公司测序，通过比对得到完整的目的基因外显子序列以及突变位点信息；
(2)gRNA设计及合成
避开突变位点，在外显子区寻找PAM序列，并在PAM序列前20bp左右设计靶序列，所有的靶序列在全基因组比对，无同源性的靶位点作为gRNA并合成；
(3)CRISPR/Cas9基因敲除载体构建
将gRNA连入基础载体VK001-02，以人源U6启动子启动gRNA的表达，T7启动子启动Cas9酶的表达，插入GFP绿色荧光基因作为报告基因，以及puro基因作为抗性药筛基因。
(4)CRISPR/Cas9基因敲除载体外源活性检测
将gRNA序列(包括PAM序列)通过搭桥PCR的方式构建到PCR产物框架中合成酶切DNA，体外转录gRNA加入反应体系，并以已知SSA活性的标准品为对照，Cas9酶体外切割反应检测luciferase信号，通过比对酶切条带灰度获得luciferase活性，luciferase活性越高则gRNA体外Cas9酶切活性越高；
(5)CRISPR/Cas9基因敲除载体内源活性检测
选择剪切活性较高CRISPR/Cas9敲除载体转染状态良好的绵羊耳成纤维细胞，24h后用嘌呤霉素筛选出阳性细胞，提取基因组DNA，克隆出靶位点前后1000bp左右片段(靶点不要位于正中间，便于分辨酶切条带)，T7E1酶进行活性检测，若存在敲除活性，将产生非配对DNA片段，即能被非配对内切酶--T7核酸内切酶I剪切；若没有发生突变，将产生配对DNA片段，而无法被非配对内切酶--T7核酸内切酶I剪切；
(6)CRISPR/Cas9基因敲除载体敲除效果检测
选择兼有外源活性和内源活性的载体，脂质体介导法转染绵羊骨骼肌卫星细胞，48h后用嘌呤霉素筛选出阳性细胞，加入增殖培养基正常培养至细胞密度达到40-50％，降低血清浓度至1％饥饿诱导卫星细胞成肌分化，同时以正常培养的细胞同期诱导作为对照。诱导72h，统计肌管形成数目。
本发明公开了一种应用CRISPR/Cas9技术对绵羊MSTN基因进行定向敲除，并验证其对骨骼肌卫星细胞分化影响效果的方法，具体内容如下：(1)载体构建：在NCBI上查询绵羊MSTN基因的CDS序列，克隆并测序以获得SNP位点，避开突变位点设计CRISPR/Cas9敲除靶位点作为gRNA，以VK001-02为基础载体构建CRISPR/Cas9双启动子敲除载体，并插入GFP蛋白作为报告基因以及puro基因作为药筛基因；(2)载体活性检测：将gRNA序列(包括PAM序列)通过搭桥PCR的方式构建到PCR产物框架中合成酶切DNA，以已知SSA活性的标准品为对照，Cas9酶切反应后比对条带灰度获取luciferase信号，选取体外Cas9酶切活性最高的gRNA进行内源活性验证；将筛选出来的载体质粒以脂质体介导法转染状态良好的绵羊耳成纤维细胞，24h后嘌呤霉素筛选阳性细胞，提取基因组DNA，克隆出靶位点前后1000bp左右片段，T7E1酶进行剪切活性检测，若存在敲除活性，将产生非配对DNA片段；(3)成肌分化验证：选择兼有外源活性和内源活性的载体，脂质体介导法转染绵羊骨骼肌卫星细胞，48h后用嘌呤霉素筛选出阳性细胞，加入增殖培养基正常培养至细胞密度达到40-50％，1％血清浓度饥饿诱导肌肉卫星细胞成肌分化，同时以正常培养的细胞同期诱导作为对照。诱导72h，间接免疫荧光染色统计肌管形成数目。
传统的ZFN技术因具有较低特异性的同源二聚体形式会切割基因组中的假回文序列，而单一ZFN单元结合于DNA也会造成DNA的剪切，这些非特异性剪切都会影响基因敲除效率，甚至可能引起细胞毒性。TALEN作为第二代EEN技术在特异性剪切方面有了很大的提高，但仍具有一定细胞毒性，且模块组装过程繁琐，需要较多人力物力。新型CRISPR/Cas9系统模仿细菌用来抵御病毒侵袭/躲避哺乳动物免疫反应的基因系统，更易于操作，效率更高，更容易得到纯合子突变体，而且可以在不同的位点同时引入多个突变，具有良好的发展前景。
本发明采用新型CRISPR/Cas9技术对绵羊MSTN基因实施定点敲除，评估敲除效率，并验证MSTN基因缺失表达对于骨骼肌卫星细胞成肌分化的影响。采用本方法，本方法简单易行，实验者可以在两个月内完成目的基因Cas9敲除载体的构建及活性验证，且靶向敲除效率明显高于传统ENN技术，MSTN基因的体外Cas9酶切活性高达90％以上。此外本方法对细胞的毒性作用很小，为后期功能性验证和转基因动物个体制备提供有力的技术支持。
本发明的优越性在于：
1、实验周期较短速；
2、方法简单易行，可重复性强，在普通实验室即可进行；
3、运用该方法构建的活性载体，毒副作用小，可用于转基因动物的制备与生产；
4、该方法非特异剪切较少，显著提高了基因靶向敲除效率高。
5、该方法适用性高，可通过敲除负调控基因用于改善各种家畜的育种性状。
附图说明
图1为本发明PCR体外扩增MSTN基因图。
图2为本发明CRISPR/Cas9-gRNA敲除载体构建图谱。
图3为本发明Cas9酶体外切割实验验证gRNA靶序列敲除活性图。
图4为本发明敲除质粒转染成纤维细胞验证内源活性图。
图5为本发明MSTN基因异位表达对骨骼肌卫星细胞成肌分化的影响图。
本材料涉及的基因序列见“说明书核苷酸和氨基酸序列表”。
具体实施方式
1.CRISPR/Cas9基因敲除载体构建
(1)目的基因克隆：查询NCBI数据库，获取绵羊目的基因的CDS序列(ID：NM_001009428)，应用Primer5.0软件设计特异性引物，以湖羊肌肉组织总RNA反转录形成的cDNA为模板PCR克隆并测序获得目的基因全部CDS序列，纯化回收后送公司测序，通过比对得到完整的目的基因外显子序列以及突变位点信息；
(2)gRNA设计及合成：避开突变位点，在外显子区寻找PAM序列，并在PAM序列前20bp左右设计靶序列，所有的靶序列在全基因组比对，无同源性的靶位点作为gRNA并合成；
(3)CRISPR/Cas9基因敲除载体构建：将合成的gRNA连入基础载体VK001-02，以人源U6启动子启动gRNA的表达，T7启动子启动Cas9酶的表达，插入GFP绿色荧光基因作为报告基因，以及puro基因作为抗性药筛基因。
2.CRISPR/Cas9基因敲除载体的活性检测
(1)外源活性检测：通过搭桥PCR的方式，将gRNA序列(包括PAM序列)构建到PCR产物框架中合成酶切DNA，体外转录gRNA加入反应体系，并以已知SSA活性的标准品为对照，Cas9酶切反应检测luciferase信号，通过比对酶切条带灰度获得luciferase活性，luciferase活性越高则gRNA体外Cas9酶切活性越高；
(2)内源活性检测：选择剪切活性较高CRISPR/Cas9敲除载体转染状态良好的绵羊耳成纤维细胞，即，选择外源活性较高CRISPR/Cas9敲除载体，使用FuGene转染试剂按转染试剂：质粒为1μl：3μg的比例转染状态良好的绵羊耳成纤维细胞，24h后用嘌呤霉素筛选出阳性细胞，提取基因组DNA，克隆出靶位点前后1000bp左右片段，靶位点不要放在正中，避免酶切条带重合。T7E1酶进行活性检测，若存在敲除活性，将产生非配对DNA片段，即能被非配对内切酶--T7核酸内切酶I剪切；若没有发生突变，将产生配对DNA片段，而无法被非配对内切酶--T7核酸内切酶I剪切；
再具体点，选择Cas9酶切活性最高CRISPR/Cas9敲除载体质粒转染状态良好的绵羊耳成纤维细胞，使用FuGene转染试剂按转染试剂：质粒为1μl：3μg的比例进行，24h观察荧光，确定转染效率，并换上含10μg/ml嘌呤霉素的培养基筛选阳性细胞，每隔24h换液，连续筛选72h后，换增殖培养基正常培养至汇合度达到70％时收集细胞，提取基因组DNA，克隆出靶位点前后1000bp左右片段，靶位点不要放在正中，避免酶切条带重合。T7E1酶进行活性检测，若存在敲除活性，将产生非配对DNA片段，即能被非配对内切酶--T7核酸内切酶I剪切；若没有发生突变，将产生配对DNA片段，而无法被非配对内切酶--T7核酸内切酶I剪切；
3.CRISPR/Cas9基因敲除载体敲除效果检测
选择兼有外源活性和内源活性的载体，脂质体介导法转染绵羊骨骼肌卫星细胞(sSMSCs)，转染48h后用工作浓度9μg/ml的嘌呤霉素筛选出阳性细胞3d后，加入增殖培养基正常培养至细胞密度达到40-50％，用含1％血清的DMEM/F12培养基诱导成肌细胞分化为肌管，降低血清浓度饥饿法诱导成肌细胞分化，同时以正常培养的细胞同期诱导作为对照。诱导72h，间接免疫荧光鉴定成肌分化标记蛋白Desmin，观察荧光，统计肌管形成数目，显著性分析数据总结MSTN基因敲除后的影响效果，为后期的转基因个体培育提供支持。
本方法应用CRISPR/Cas9技术对绵羊MSTN基因进行定向敲除，并验证其对骨骼肌卫星细胞分化效果的影响。首先克隆出绵羊MSTN基因的CDS序列，测序并比对序列以获得SNP位点，避开突变位点设计CRISPR/Cas9敲除靶位点作为gRNA，以VK001-02为基础载体改建CRISPR/Cas9双启动子敲除载体，并插入GFP蛋白作为报告基因以及puro基因作为药筛基因；然后对载体进行外源和内源活性检测，外源活性检测使用Cas9酶对包含gRNA(包括PAM序列)的DNA序列进行酶切实验，以已知SSA活性的标准品为对照，通过比对条带灰度获取luciferase信号，选取体外Cas9酶切活性最高的gRNA载体质粒以脂质体介导法转染绵羊耳成纤维细胞进行内源活性验证，24h后嘌呤霉素筛选阳性细胞，提取基因组DNA，克隆出靶位点前后1000bp左右片段，T7E1酶进行剪切活性检测；最后，筛选体内外活性较高的载体进行成肌分化的验证，脂质体介导法转染绵羊骨骼肌卫星细胞，48h后用嘌呤霉素筛选出阳性细胞，加入增殖培养基正常培养至细胞密度达到70％，1％血清浓度饥饿法诱导卫星细胞成肌分化，同时以正常培养的细胞同期诱导作为对照。诱导72h，间接免疫荧光染色统计肌管形成数目。最终通过统计分析结果总结出MSTN基因敲除后影响成肌细胞分化的效果。
附图中，图1为本发明PCR体外扩增MSTN基因图，扩增片段大小为1309bp。
图2为本发明CRISPR/Cas9-gRNA敲除载体构建图谱。
图3为本发明Cas9酶体外切割实验验证gRNA靶序列敲除活性图。NC为对照组，1、2、3、4分别为预设计的四个的gRNA靶点，标g1和标g2分别为经过SSAluciferase检测活性为3和10的标准品，以标准品为参照比对条带灰度获得四个靶点的酶切活性，结果见表1。
表1Cas9酶体外切割靶位点活性检测
| 样品名字 | 靶序列(灰色区域为PAM序列) | 体外酶切活性 |
| MSTN-g1 | ATTTATGCTGCTTGTTGCTGG | 50％ |
| MSTN-g4 | TAAGACAACTTTTGCCCAAGG | 65％ |
| MSTN-g6 | AACACAATAAAGTAGTAAAGG | 5％ |
| MSTN-g7 | ATGGGTTTGATGAGTCTCAGG | 95％ |
图4敲除质粒转染成纤维细胞验证内源活性：A组分别为荧光暗场、明暗交界、明场条件下观察细胞转染效果；图B为T7E1酶切效果，W为野生型，M为突变型。
图5MSTN基因异位表达对骨骼肌卫星细胞成肌分化的影响：间接免疫荧光检测成肌分化早期标志Desmin蛋白(100×)，结果显示敲除组(Treatment)肌管细胞形成数目显著高于对照组(Control)。
SEQUENCELISTING
<110>扬州大学
<120>一种绵羊MSTN基因定向敲除及其影响成肌分化的检测方法
<130>2015
<160>1
<170>PatentInversion3.3
<210>1
<211>1128
<212>DNA
<213>Ovisaries(MyostatinGene)
<400>1
atgcaaaaactgcaaatctttgtttatatttacctatttatgctgcttgttgctggccca60
gtggatctgaatgagaacagcgagcagaaggaaaatgtggaaaaaaaggggctgtgtaat120
gcatgcttgtggagacaaaacaataaatcctcaagactagaagccataaaaatccaaatc180
ctcagtaagcttcgcctggaaacagctcctaacatcagcaaagatgctataagacaactt240
ttgcccaaggctcctccactccgggaactgattgatcagtacgatgtccagagagatgac300
agcagcgacggctccttggaagacgatgactaccacgttacgacggaaacggtcattacc360
atgcccacggagtctgatcttctagcagaagtgcaagaaaaacccaaatgttgcttcttt420
aaatttagctctaagatacaacacaataaagtagtaaaggcccaactgtggatatatctg480
agacctgtcaagactcctacaacagtgtttgtgcaaatcctgagactcatcaaacccatg540
aaagacggtacaaggtatactggaatccgatctctgaaacttgacatgaacccaggcact600
ggtatttggcagagcattgatgtgaagacagtgttgcaaaactggctcaaacaacctgaa660
tccaacttaggcattgaaatcaaagctttagatgagaatggtcatgatcttgctgtaacc720
ttcccagaaccaggagaagaaggactgaatccttttttagaagtcaaggtaacagacaca780
ccaaaaagatctaggagagattttgggcttgattgtgatgagcactccacagaatctcga840
tgctgtcgttaccctctaactgtggattttgaagcttttggatgggattggattattgca900
cctaaaagatataaggccaattactgctctggagaatgtgaatttttatttttgcaaaag960
tatcctcatacccatcttgtgcaccaagcaaaccccaaaggttcagccggcccttgctgt1020
actcctacaaagatgtctccaattaatatgctatattttaatggcaaagaacaaataata1080
tatgggaagattccaggcatggtagtagatcgctgtgggtgctcatga1128
Claims (2)
1.一种绵羊MSTN基因定向敲除及其影响成肌分化的检测方法，其特征在于，包括如下步骤：
(1)目的基因克隆
查询NCBI数据库，获取绵羊目的基因的CDS序列，绵羊目的基因的CDS序列的序列号为NM_001009428，设计特异性引物，以湖羊肌肉组织总RNA反转录形成的cDNA为模板克隆并获得目的基因全部CDS序列，纯化回收后送公司测序，通过比对得到完整的目的基因外显子序列以及突变位点信息；
(2)gRNA设计及合成
避开突变位点，在外显子区寻找PAM序列，并在PAM序列前20bp左右设计靶序列，所有的靶序列在全基因组比对，无同源性的靶位点作为gRNA并合成；
(3)CRISPR/Cas9基因敲除载体构建
将gRNA连入基础载体VK001-02，以人源U6启动子启动gRNA的表达，T7启动子启动Cas9酶的表达，插入GFP绿色荧光基因作为报告基因，以及puro基因作为抗性药筛基因；
(4)CRISPR/Cas9基因敲除载体外源活性检测
将gRNA序列，包括PAM序列，通过搭桥PCR的方式构建到PCR产物框架中合成酶切DNA，体外转录gRNA加入反应体系，并以已知SSA活性的标准品为对照，Cas9酶切反应检测luciferase信号，通过比对酶切条带灰度获得luciferase活性，luciferase活性越高则gRNA体外Cas9酶切活性越高；
(5)CRISPR/Cas9基因敲除载体内源活性检测
选择剪切活性较高CRISPR/Cas9敲除载体转染状态良好的绵羊耳成纤维细胞，24h后用嘌呤霉素筛选出阳性细胞，提取基因组DNA，克隆出靶位点前后1000bp左右片段，靶位点不要放在正中，避免酶切条带重合；T7E1酶进行活性检测，若存在敲除活性，将产生非配对DNA片段，即能被非配对内切酶--T7核酸内切酶I剪切；若没有发生突变，将产生配对DNA片段，而无法被非配对内切酶--T7核酸内切酶I剪切；
(6)CRISPR/Cas9基因敲除载体敲除效果检测
选择兼有外源活性和内源活性的载体，脂质体介导法转染绵羊骨骼肌卫星细胞，48h后用嘌呤霉素连续筛选72h，加入增殖培养基正常培养至细胞密度达到40-50％，1％血清浓度饥饿法诱导卫星细胞成肌分化，同时以正常培养的细胞同期诱导作为对照，诱导72h，间接免疫荧光染色统计肌管形成数目。
2.根据权利要求1所述的一种绵羊MSTN基因定向敲除及其影响成肌分化的检测方法，其特征是，步骤(1)中，应用Primer5.0软件设计特异性引物。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610237236.6A CN105821116A (zh) | 2016-04-15 | 2016-04-15 | 一种绵羊mstn基因定向敲除及其影响成肌分化的检测方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610237236.6A CN105821116A (zh) | 2016-04-15 | 2016-04-15 | 一种绵羊mstn基因定向敲除及其影响成肌分化的检测方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105821116A true CN105821116A (zh) | 2016-08-03 |
Family
ID=56526022
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610237236.6A Pending CN105821116A (zh) | 2016-04-15 | 2016-04-15 | 一种绵羊mstn基因定向敲除及其影响成肌分化的检测方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105821116A (zh) |
Cited By (45)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106636002A (zh) * | 2017-01-13 | 2017-05-10 | 湖北省农业科学院畜牧兽医研究所 | Mstn双敲细胞系及其构建方法 |
| CN106947780A (zh) * | 2017-03-28 | 2017-07-14 | 扬州大学 | 一种兔mstn基因的编辑方法 |
| CN106957857A (zh) * | 2016-09-23 | 2017-07-18 | 西北农林科技大学 | 一种利用CRISPR/Cas9系统共同敲除山羊MSTN和FGF5基因的方法 |
| CN107034221A (zh) * | 2017-06-02 | 2017-08-11 | 内蒙古大学 | 一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因及应用 |
| CN107164527A (zh) * | 2017-06-29 | 2017-09-15 | 杭州观梓健康科技有限公司 | 一种筛选参与多能干细胞体外定向分化调节因子的方法 |
| US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
| CN107868798A (zh) * | 2017-03-31 | 2018-04-03 | 上海市公共卫生临床中心 | 一种基于基因敲除细胞的阳性筛选体系的建立方法 |
| CN108018311A (zh) * | 2016-11-04 | 2018-05-11 | 中国科学院上海生命科学研究院 | 通过基因编辑特异靶向肌肉组织mstn治疗恶病质 |
| CN108048396A (zh) * | 2017-12-29 | 2018-05-18 | 山西农业大学 | 一种绵羊肌肉干细胞的分离方法 |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| CN110305910A (zh) * | 2019-07-17 | 2019-10-08 | 吴忠市红寺堡区天源农牧业科技开发有限公司 | 利用CRISPR/Cas9系统大片段敲除绵羊MSTN基因的方法 |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| CN111778289A (zh) * | 2020-07-27 | 2020-10-16 | 扬州大学 | 一种利用CRISPR-Cas9靶向敲除鸡Bmp4基因的方法 |
| CN111849987A (zh) * | 2020-07-29 | 2020-10-30 | 华农（肇庆）生物产业技术研究院有限公司 | Viperin功能缺失的细胞系的构建方法及其应用 |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| CN114438232A (zh) * | 2022-03-11 | 2022-05-06 | 浙江省农业科学院 | SNPs分子标记g.43917 A＞G及其在湖羊分子标记辅助育种中的应用 |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| 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 |
| CN118147229A (zh) * | 2024-04-01 | 2024-06-07 | 内蒙古大学 | 一种新型Cas13a/cr:mi:crRNA基因敲减工具、构建方法及其应用 |
| CN118497232A (zh) * | 2024-04-16 | 2024-08-16 | 西北农林科技大学 | 一种融合核酸外切酶的CRISPR/Cas12i基因编辑衍生系统及其应用 |
| US12157760B2 (en) | 2018-05-23 | 2024-12-03 | The Broad Institute, Inc. | Base editors and uses thereof |
| US12281338B2 (en) | 2018-10-29 | 2025-04-22 | The Broad Institute, Inc. | Nucleobase editors comprising GeoCas9 and uses thereof |
| US12351837B2 (en) | 2019-01-23 | 2025-07-08 | The Broad Institute, Inc. | Supernegatively charged proteins and uses thereof |
| US12390514B2 (en) | 2017-03-09 | 2025-08-19 | President And Fellows Of Harvard College | Cancer vaccine |
| US12406749B2 (en) | 2017-12-15 | 2025-09-02 | The Broad Institute, Inc. | Systems and methods for predicting repair outcomes in genetic engineering |
| US12435330B2 (en) | 2019-10-10 | 2025-10-07 | The Broad Institute, Inc. | Methods and compositions for prime editing RNA |
| US12473543B2 (en) | 2019-04-17 | 2025-11-18 | The Broad Institute, Inc. | Adenine base editors with reduced off-target effects |
| US12522807B2 (en) | 2018-07-09 | 2026-01-13 | The Broad Institute, Inc. | RNA programmable epigenetic RNA modifiers and uses thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290045A (zh) * | 2013-04-26 | 2013-09-11 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | 利用锌指核酸酶敲除绵羊肌肉抑制素基因的方法 |
| CN103952405A (zh) * | 2014-05-13 | 2014-07-30 | 扬州大学 | 一种山羊mstn基因定点修饰系统及其应用 |
| CN104805118A (zh) * | 2015-04-22 | 2015-07-29 | 扬州大学 | 一种苏禽黄鸡胚胎干细胞特定基因进行靶向敲除方法 |
| CN105132427A (zh) * | 2015-09-21 | 2015-12-09 | 新疆畜牧科学院生物技术研究所 | 一种以RNA介导的特异性敲除双基因获得基因编辑绵羊的方法及其专用sgRNA |
-
2016
- 2016-04-15 CN CN201610237236.6A patent/CN105821116A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103290045A (zh) * | 2013-04-26 | 2013-09-11 | 新疆维吾尔自治区畜牧科学院中国-澳大利亚绵羊育种研究中心 | 利用锌指核酸酶敲除绵羊肌肉抑制素基因的方法 |
| CN103952405A (zh) * | 2014-05-13 | 2014-07-30 | 扬州大学 | 一种山羊mstn基因定点修饰系统及其应用 |
| CN104805118A (zh) * | 2015-04-22 | 2015-07-29 | 扬州大学 | 一种苏禽黄鸡胚胎干细胞特定基因进行靶向敲除方法 |
| CN105132427A (zh) * | 2015-09-21 | 2015-12-09 | 新疆畜牧科学院生物技术研究所 | 一种以RNA介导的特异性敲除双基因获得基因编辑绵羊的方法及其专用sgRNA |
Non-Patent Citations (1)
| Title |
|---|
| 李聪: "CRISPR/Cas9技术对绵羊肌肉卫星细胞MSTN基因的靶向敲除研究", 《中国优秀硕士学位论文全文数据库农业科技辑》 * |
Cited By (78)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US12473573B2 (en) | 2013-09-06 | 2025-11-18 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US9840699B2 (en) | 2013-12-12 | 2017-12-12 | President And Fellows Of Harvard College | Methods for nucleic acid editing |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US12215365B2 (en) | 2013-12-12 | 2025-02-04 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US12398406B2 (en) | 2014-07-30 | 2025-08-26 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US12043852B2 (en) | 2015-10-23 | 2024-07-23 | President And Fellows Of Harvard College | Evolved Cas9 proteins for gene editing |
| US12344869B2 (en) | 2015-10-23 | 2025-07-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11999947B2 (en) | 2016-08-03 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US12084663B2 (en) | 2016-08-24 | 2024-09-10 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| CN106957857A (zh) * | 2016-09-23 | 2017-07-18 | 西北农林科技大学 | 一种利用CRISPR/Cas9系统共同敲除山羊MSTN和FGF5基因的方法 |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| CN108018311A (zh) * | 2016-11-04 | 2018-05-11 | 中国科学院上海生命科学研究院 | 通过基因编辑特异靶向肌肉组织mstn治疗恶病质 |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| CN106636002A (zh) * | 2017-01-13 | 2017-05-10 | 湖北省农业科学院畜牧兽医研究所 | Mstn双敲细胞系及其构建方法 |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US12516308B2 (en) | 2017-03-09 | 2026-01-06 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US12390514B2 (en) | 2017-03-09 | 2025-08-19 | President And Fellows Of Harvard College | Cancer vaccine |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US12435331B2 (en) | 2017-03-10 | 2025-10-07 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| CN106947780A (zh) * | 2017-03-28 | 2017-07-14 | 扬州大学 | 一种兔mstn基因的编辑方法 |
| CN107868798A (zh) * | 2017-03-31 | 2018-04-03 | 上海市公共卫生临床中心 | 一种基于基因敲除细胞的阳性筛选体系的建立方法 |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| CN107034221A (zh) * | 2017-06-02 | 2017-08-11 | 内蒙古大学 | 一种能在小鼠体内表达的部分碱基缺失肌肉抑制素基因及应用 |
| CN107164527A (zh) * | 2017-06-29 | 2017-09-15 | 杭州观梓健康科技有限公司 | 一种筛选参与多能干细胞体外定向分化调节因子的方法 |
| CN107164527B (zh) * | 2017-06-29 | 2018-04-20 | 杭州观梓健康科技有限公司 | 一种筛选参与多能干细胞体外定向分化调节因子的方法 |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US12359218B2 (en) | 2017-07-28 | 2025-07-15 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US12406749B2 (en) | 2017-12-15 | 2025-09-02 | The Broad Institute, Inc. | Systems and methods for predicting repair outcomes in genetic engineering |
| CN108048396A (zh) * | 2017-12-29 | 2018-05-18 | 山西农业大学 | 一种绵羊肌肉干细胞的分离方法 |
| US12157760B2 (en) | 2018-05-23 | 2024-12-03 | The Broad Institute, Inc. | Base editors and uses thereof |
| US12522807B2 (en) | 2018-07-09 | 2026-01-13 | The Broad Institute, Inc. | RNA programmable epigenetic RNA modifiers and uses thereof |
| US12281338B2 (en) | 2018-10-29 | 2025-04-22 | The Broad Institute, Inc. | Nucleobase editors comprising GeoCas9 and uses thereof |
| US12351837B2 (en) | 2019-01-23 | 2025-07-08 | The Broad Institute, Inc. | Supernegatively charged proteins and uses thereof |
| US12281303B2 (en) | 2019-03-19 | 2025-04-22 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US12509680B2 (en) | 2019-03-19 | 2025-12-30 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | 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 |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US12473543B2 (en) | 2019-04-17 | 2025-11-18 | The Broad Institute, Inc. | Adenine base editors with reduced off-target effects |
| CN110305910A (zh) * | 2019-07-17 | 2019-10-08 | 吴忠市红寺堡区天源农牧业科技开发有限公司 | 利用CRISPR/Cas9系统大片段敲除绵羊MSTN基因的方法 |
| US12435330B2 (en) | 2019-10-10 | 2025-10-07 | The Broad Institute, Inc. | Methods and compositions for prime editing RNA |
| US12031126B2 (en) | 2020-05-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| 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 |
| CN111778289A (zh) * | 2020-07-27 | 2020-10-16 | 扬州大学 | 一种利用CRISPR-Cas9靶向敲除鸡Bmp4基因的方法 |
| CN111849987A (zh) * | 2020-07-29 | 2020-10-30 | 华农（肇庆）生物产业技术研究院有限公司 | Viperin功能缺失的细胞系的构建方法及其应用 |
| CN114438232A (zh) * | 2022-03-11 | 2022-05-06 | 浙江省农业科学院 | SNPs分子标记g.43917 A＞G及其在湖羊分子标记辅助育种中的应用 |
| CN118147229A (zh) * | 2024-04-01 | 2024-06-07 | 内蒙古大学 | 一种新型Cas13a/cr:mi:crRNA基因敲减工具、构建方法及其应用 |
| CN118147229B (zh) * | 2024-04-01 | 2025-04-29 | 内蒙古大学 | 一种新型Cas13a/cr:mi:crRNA基因敲减工具、构建方法及其应用 |
| CN118497232A (zh) * | 2024-04-16 | 2024-08-16 | 西北农林科技大学 | 一种融合核酸外切酶的CRISPR/Cas12i基因编辑衍生系统及其应用 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105821116A (zh) | 一种绵羊mstn基因定向敲除及其影响成肌分化的检测方法 | |
| US11317610B2 (en) | Method of constructing zebrafish notch1a mutants | |
| Liang et al. | Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection | |
| US20230406893A1 (en) | Pam restriction-free adenine base editor fused protein and use thereof | |
| EP3536796A1 (en) | Gene knockout method | |
| WO2017190664A1 (zh) | 化学合成的crRNA和修饰crRNA在CRISPR/Cpf1基因编辑系统中的应用 | |
| CN103388006A (zh) | 一种基因定点突变的构建方法 | |
| EP3940078A1 (en) | Off-target single nucleotide variants caused by single-base editing and high-specificity off-target-free single-base gene editing tool | |
| CN109880851A (zh) | 用于富集CRISPR/Cas9介导的同源重组修复细胞的筛选报告载体及筛选方法 | |
| CN106086031A (zh) | 猪肌抑素基因编辑位点及其应用 | |
| CN108949832A (zh) | 一种用于敲除猪ghr基因的打靶载体及其应用 | |
| CN107267516B (zh) | 双sgRNA介导的基因精确修饰方法及应用 | |
| CN115786304A (zh) | 一种Cas12a蛋白突变体、含有其的碱基编辑器和应用 | |
| Lu et al. | CRISPR/Cas9-mediated hitchhike expression of functional shRNAs at the porcine miR-17-92 cluster | |
| US11946163B2 (en) | Methods for measuring and improving CRISPR reagent function | |
| Tian et al. | Targeted G-to-T base editing for generation of novel herbicide-resistance gene alleles in rice | |
| CN119307495A (zh) | 特异性靶向猪HMGA1和HMGA2基因的sgRNA导向序列及其应用 | |
| CN116064541B (zh) | 一种基于CRISPR/Cas9系统敲除猪细胞Y染色体的方法 | |
| Wu et al. | CRISPR/Cas9 (D10A) nickase-mediated Hb CS gene editing and genetically modified fibroblast identification | |
| CN113881703B (zh) | 一种提高cho细胞同源重组效率的方法及其相关产品和应用 | |
| CN113403342A (zh) | 一种单碱基突变方法及采用的系统 | |
| CN104894255A (zh) | 一种基于聚丙烯酰胺凝胶电泳检测低效率基因组编辑的方法及其应用 | |
| CN111088253A (zh) | 针对hbb-28地中海贫血基因的crispr单碱基供体修复体系 | |
| CN110408621A (zh) | 猕猴rosa26基因及其基因修饰的方法和应用 | |
| CN114875033A (zh) | sgRNA、CRISPR/Cas试剂及其应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160803 |
|
| RJ01 | Rejection of invention patent application after publication |